Role of oxidants

COMENTARIOS CLÍNICOS microbiología, 0893-8512/97 / 04,0010 dólares Enero 1997, p. Vol. 1-18. 10, No. 1 Copyright q de 1997, la Sociedad Americana de Microbiología El papel de los oxidantes en Fisiopatología microbiana RACHEL * A. MILLER y Bradley E. BRITIGAN División de Enfermedades Infecciosas del Departamento de Medicina Interna, Administración de Veteranos del Centro Médico, y la Universidad de Iowa College of Medicine, Iowa City, Iowa 52242

INTRODUCCIÓN ........................ 1


* Generación y toxicidad de seleccionados oxidantes en sistemas biológicos.. 1


* Superóxido y peróxido de hidrógeno ....... 2


* El radical hidroxilo ......... .... 2


* Fuentes de hierro disponible para la generación de radicales hidroxilo en Vivo ................... 3


* Oxidantes mieloperoxidasa Derivados ........... 3


* El óxido nítrico ................... 3


* FUENTES de oxidantes encontrados por los microbios en VIVO ............... 4


* Fuentes endógenas ................................. 4


* Las fuentes exógenas .............................. 4


* oxidantes derivados de los fagocitos y su papel en la defensa del huésped ..................... 4


* Otras fuentes de antioxidantes y su contribución a la actividad microbicida ............ 6


* MECANISMOS DE DEFENSA MICROBIANA contra los oxidantes ............... 7


* Prevención de Encuentros con fagocitos Derivados Oxidantes .................. 7


* Estrategias de defensa concreta de los oxidantes ....................... 7


* No enzimática .......................................... .... 7


* Enzimática ...................................................... 8


* PAPEL de oxidantes en las infecciones víricas ............................. 9


* Indeseables consecuencias de la producción OXIDANTE PARA EL ANFITRIÓN ............. 10


* CONCLUSIONES ................................. ..... 12


* AGRADECIMIENTOS ........................... 12


* REFERENCIAS ............................... 12


INTRODUCCIÓN Las especies reactivas de oxígeno han sido cada vez más implicados desempeña un papel central en la fisiopatología de la clínica infecciones. Más concretamente, el superóxido, el peróxido de hidrógeno hidroxilo del ácido radical, hypohalous, y recientemente, el óxido nítrico son cree que contribuyen a estos procesos. Estos compuestos exhiben un amplio espectro de biotoxicidad y son cruciales para acoger defensa de la actividad óptima microbicida de los neutrófilos y otros fagocitos (148, 209, 216, 313). En respuesta, los microorganismos han desarrollado estrategias complejas no sólo para evitar contacto con oxidantes derivados de los fagocitos, sino también para defender a sí mismos de lesiones una vez oxidantes se encuentran. Anfitrión las células han desarrollado adaptaciones similares para protegerse en contra de una consecuencia nociva de la exposición oxidante, inflamatoria lesiones en los tejidos (209, 313).

En esta revisión se discuten los formación de oxidantes in vivo y su papel central en la compleja interacción entre la invasión microbiana y la defensa del huésped. GENERACIÓN Y TOXICIDAD DE SELECCIONADOS Oxidantes en los sistemas biológicos Muchas reacciones bioquímicas vitales para el metabolismo aeróbico normal de las células humanas y microbianos exigir la transferencia de cuatro electrones al oxígeno molecular para formar H2O.

En la mayoría de circunstancias, esta transferencia se produce de forma simultánea sin que el formación de otros intermediarios. Sin embargo, el oxígeno molecular tiene la capacidad de someterse a la reducción univalente secuencial para formar otros intermediarios de oxígeno con diferentes toxicidades antes de la generación de H2O. La adición de un electrón a los rendimientos de la superóxido O2 radical (ZO2 2), que a pH fisiológico se reduce rápidamente (Dismutes, k '2 3 105 M21 s21) para formar el oxígeno divalente producto de reducción, el peróxido de hidrógeno (H2O2). Trivalente oxígeno in vitro se produce a través de la reducción de la reacción del H2O2 con ZO2 2 para producir el radical hidroxilo (zoH). Sin embargo, al fisiológica pH, esta reacción es de importancia biológica poco menos que un catalizador de metales de transición (por ejemplo, Fe31) está presente para mejorar la velocidad de reacción, dando zoH a través de la reacción de Haber-Weiss (123) (Tabla 1). Como se señalaba anteriormente, no todos los complejos de hierro puede servir como catalizador de esta reacción (125).

Además de la formación zoH, inducida experimentalmente las interacciones entre H2O2 y el hierro quelatos también puede conducir a la producción de la plancha reactiva peroxocomplex y el ion ferryl (268, 321). Sin embargo, su papel en fisiología humana y microbiana es en gran parte desconocida. Aunque la mayoría de las investigaciones se han centrado en la formación de zoH a través del mecanismo de Haber-Weiss, la evidencia también existe para la formación de zoH de ZO2 reducción de 2-mediada de hipocloroso ácido (HOCl) (51, 189, 232, 250). Un oxidante potente en sí mismo, HOCl se genera por la interacción de H2O2 con peroxidasas fagocito derivados (148). Recientemente, una intensa investigación se ha dirigido a otra especies oxidantes, óxido nítrico (NOZ).

Noz no es un producto clásico de la reducción de O2, en cambio, su formación en células de mamíferos es depende de un grupo de enzimas denominado sintasa de óxido nítrico (NOS) (216, 224). Estas enzimas oxidan L-arginina para L-citrulina y Noz. Aunque varias isoformas de NOS relacionados Se han aislado, se dividen en dos categorías, constitutiva inducible y, basándose en las diferencias en la regulación y las actividades. isoformas constitutivas (CNOS) se encuentran en las neuronas * Autor para la correspondencia. Dirección postal: Departamento de Interior Medicina de la Universidad de Iowa, el Dr. Hawkins 200, SW 34 GH, Iowa City, Iowa 52242. Teléfono: (319) 356-7228. Fax: (319) 356-4600. 1 Descargar de cmr.ASM.ORG - 20 de julio 2010 y las células endoteliales. CNOS actividad responde a los cambios en la concentración de calcio intracelular a través de calcio-calmodulina vinculante. Esto resulta en la producción intermitente de las pequeñas cantidades de Noz necesarios para los procesos fisiológicos tales como neurotransmisión y la regulación de la presión arterial. Mediante el uso de técnicas derivadas de captura, NOS cerebro también se ha demostrado para generar ZO2 2 en un calcio-calmodulina-dependiente forma (243). La isoforma NOS inducible (iNOS) se expresa en muchos tipos celulares, incluyendo hepatocitos, epitelio respiratorio, y los macrófagos. Su actividad es independiente de las fluctuaciones en la concentración intracelular de calcio. Los factores conocidos para modular los niveles de iNOS incluir un número de citoquinas, microorganismos, y productos microbianos, en consonancia con la importancia de la actividad de iNOS en la defensa del huésped y la inflamación. Muchas especies de bacterias son también capaces de generar Noz en condiciones de baja tensión de oxígeno a través de nitrito reductasas (334). Una vez formado, Noz tiene la capacidad de actuar como un oxidante agentes solos o interactuar con ZO2 2 para generar peroxinitrito (ONOO2) (k '6.7 3 109 M21 S21) (245, 247, 284) y en última instancia, a través de la formación zoH peroxinitrato y descomposición (Tabla 1) (19). A pesar de un catalizador de metal de transición no es necesarios en este sistema, las consideraciones termodinámicas y cinéticas no puede favorecer la formación de zoH a través de esta reacción en vivo (170). Superóxido y peróxido de hidrógeno El superóxido es un compuesto moderadamente reactivo capaz de actúa como oxidante o reductor en los sistemas biológicos. Esta relativa inactividad permite ZO2 2 para difundir a distancias considerables antes de que ejerce sus efectos tóxicos. Extracelularmente generados ZO2 2 puede ganar acceso a las intracelular a través de celulares canales de aniones (264). Estos objetivos son: las enzimas bacterianas, particular los que participan en la biosíntesis de aminoácidos ramificados ácidos (por ejemplo, a, b-deshidratasa dihydroxyisovalerate y NADHbound deshidrogenasa láctica) (122, 174). Varios coli coli (y mamíferos) que contiene deshidratasas [4Fe-4S] agrupaciones.

Son particularmente susceptibles a la inactivación por ZO2 2, incluidas las aconitasa, 6-fosfogluconato deshidratasa, a, b-dihydroxyacid deshidratasa, y fumarases A y B (101, 108, 109, 111, 183). Aconitasa También se ha demostrado que se inactiva por ONOO2 pero no Noz (57, 139). Estas enzimas son únicos en que posteriormente pueden someterse a una reactivación por irondependent mecanismo (110). Se postula que la inactivación se produce en una fase temprana del estrés oxidativo, de manera que aconitases funcionan como interruptores automáticos, "abandono de la producción de sustancias tóxicas ZO2 2 por el cierre temporal oxidativo celular metabolismo (109).

Una vez que el estrés ha pasado, el deshidratasas puede ser reactivado por el hierro intracelular y tioles en lugar de tener que ser sintetizados de novo (109). En los ambientes de pH bajo, como en los sitios de la inflamación o en el interior del fagosoma, ZO2 2 pasa a ser protonado para formar HO2 z. Debido a su carga neutra, HO2 z es la membrana más permeables y más propensos a reaccionar con sí mismo para formar H2O2. adicionales de toxicidad ZO2 2 en los sistemas biológicos es probable que se produzca a través de su participación en la reacción de Haber-Weiss en la presencia de hierro catalíticamente activas (123). El peróxido de hidrógeno es un oxidante más reactivo que ZO2 2, y fácilmente difunde a través de las membranas celulares. Las fuentes potenciales de daños H2O2-mediada de los componentes celulares incluyen la la oxidación de las membranas celulares y las enzimas, daño en el ADN y mutagénesis, y la inhibición del transporte de membrana procesos (313). Imlay y Linn han descrito con mayor detalles de los mecanismos de daño mediado por H2O2.

Demostraron que la matanza de E. coli por producción de H2O2 es bimodal en el que los bajos (1 a 3 mm) y alto (0,20 mM) son las concentraciones de H2O2 más letales que las concentraciones intermedias (153). Modo 1 (Concentración baja H2O2) asesinato ha sido atribuido al ADN daño mediado por la interacción de H2O2 con Fe21 a formar el radical tóxicos ferryl (149), un producto intermedio en la formación de zoH.

La exposición de E. coli a estas bajas concentraciones de H2O2 induce una respuesta protectora que confiere mayor resistencia a las posteriores exposiciones H2O2 por un mayor capacidad para llevar a cabo la reparación del ADN por recombinación (154). Modo 2 matar, que no requiere de hierro o un electrón fuente de que se produzca, no se debe a daños en el ADN, pero puede involucrar a la oxidación de un objetivo específico celular (137). Reciente datos por parte de Pacelli et al. demostrar que Noz potencia H2O2- inducida por asesinato de E. coli (235). Esto sugiere que macrophagederived Noz, además de sus efectos citotóxicos propia, pueden interferir con H2O2 para mejorar la actividad microbicida en los sitios de infección (235). El radical hidroxilo En muchos casos en ZO2 2 y / o H2O2 está implicada en la celda lesiones, no está claro si el proceso está mediado por estos compuestos o si simplemente sirven como precursores para la otra, las especies oxidantes más potentes (por ejemplo, zoH), que es verdaderamente mediación de la lesión. Los estudios con más sensible de los radicales libres sistemas de detección de implicar a zoH en la oxidación de un gran número de biomoléculas como proteínas, ADN y los lípidos, como resultado de su exposición inicial a ZO2 2 y H2O2 / o. Debido a su alta reactividad, zoH es limitada difusión de tal manera que una vez formado en un sistema biológico, es probable que viaje sólo a muy corto distancias antes de que encuentre un sustrato oxidable. Esta dicta que la propiedad zoH se debe generar en las proximidades a una molécula celular objetivo fundamental a fin de que la mediación lesión directa (77). Un mecanismo por el cual zoH y otros oxidantes puede causar lesión de las células en sitios distantes de su formación es a través de la apertura de una cascada de radicales libres (43, 313). Oxidación de los insaturados ácidos grasos dentro de una membrana lipídica puede producir peroxil radical, que a su vez puede reaccionar con otras moléculas de lípidos cercanos de generar radicales adicionales de lípidos. Estas nuevas presentaciones lipídicas a continuación, los radicales pueden reaccionar con otros lípidos insaturados, con lo que la creación de una reacción en cadena de radicales libres (43).

Esta reacción TABLA 1.

Las reacciones químicas que afectan a especies reactivas del oxígeno Reacción Formulaa Haber-Weiss reacción .............. ZO2 2 1 1 3 O2 Fe31 Fe21 H2O2 Fe21 3 1 1 zoH OH2 1 Fe31 ZO2 2 1 3 H2O2 zoH 1 OH2 1 O2 Mielodepresión (eosinófilos) peroxidasa ............................. 1 H2O2 HX 3 HOX 1 H2O Nítrico sintetasa de óxido ................ L arginina-3 L-citrulina 1 Noz la formación de peroxinitrito / descomposición ....................... Noz 1 ZO2 2 3 ONOO2 ONOO2 1 H1 3 ONOOH ONOOH 3 zoH 1 Noz GSH GSH peroxidasa ............................. 2 1 3 H2O2 GSSG 1 2H2O Reductasa .............................. GSSG 2NADPH 1 3 1 2GSH 2NADP Catalasa ..................................... 2H2O2 3 1 O2 2H2O SOD ........................................... 2zO2 2 1 3 2H1 H2O2 1 O2 una X, de halogenuros. 2 MILLER Y CLIN BRITIGAN. Microbiol. REV. Descargar de cmr.ASM.ORG - 20 de julio 2010 eventualmente se traduce en la oxidación de las biomoléculas en los sitios considerablemente distante de donde la primera reacción de los radicales libres se produjo (43). Fuentes de hierro disponible para el radical hidroxilo Generación En Vivo Desde la formación de zoH ZO2 2 y H2O2 en condiciones fisiológicas condiciones requiere la presencia de un catalizador de metal de transición, ha habido un interés considerable en la determinación de que el hierro quelatos potencialmente presente en vivo podría servir como catalizadores zoH. En los seres humanos, el hierro intracelular es predominantemente un complejo a la ferritina en una forma relativamente no catalítico (301).

Del mismo modo, casi todo el hierro extracelular está fuertemente unido al de acogida vinculante proteínas (lactoferrina y transferrina), en formas no puede catalizar zoH formación (7, 9, 42, 45, 320). De hecho, hay fuertes datos que sugieren que la lactoferrina actúa como un (antioxidante 34, 39, 40, 71, 215). lactoferrina neutrófilos pueden funcionar para atrapar el hierro de microorganismos ingeridos (215). En los fagocitos que no contienen lactoferrina (es decir, los monocitos y macrófagos), una específica receptor de la superficie se une lactoferrina exógenos (20, 24, 50, 213, 326). Los monocitos / macrófagos previamente incubadas con lactoferrina son menos susceptibles a la peroxidación de hierro-dependiente de sus membranas (40).

Así, a través de su interacción con los fagocitos, lactoferrina puede prevenir la formación de oxidantes de hierro-catalizada, limitando así el daño tisular inflamatoria. Esto complementar la capacidad de la proteína para limitar la disponibilidad de hierro para el crecimiento microbiano (88, 99). La lactoferrina también se une lipopolisacárido, un compuesto importante mediador de toxicidad en sepsis. Aunque esta interacción no tiene ningún efecto sobre la capacidad de lactoferrina para inhibir la reacción de Haber-Weiss, que se perturbe lipopolisacárido de cebado de los fagocitos para ZO2 2 Producción (71). Por el contrario, los datos in vitro sugieren que la modificación de algunos acogida quelantes del hierro o proteínas por las proteasas ZO2 2 puede dar lugar a la generación de productos capaces de catalizar la formación de zoH (23, 33).

La Pseudomonas aeruginosa producto de secreción Pseudomonas elastasa y otras proteasas derivadas de acogida presentes en los sitios de inflamación se sabe que unirá la transferrina y lactoferrina en un menor peso molecular quelatos de hierro (27, 32, 84, 90, 91, 254). Pseudomonas elastasa hendidas transferrina y, en menor medida, la lactoferrina es capaz de catalizar la formación de zoH cuando una fuente de ZO2 2 y H2O2 es al mismo tiempo presente (33, 208, 211). Estudios adicionales han demostrado la capacidad de la elastasa y otras Pseudomonas transferrina proteasa hendidas para mejorar porcina oxidante mediada arteria pulmonar lesiones de las células endoteliales a través de la generación de zoH en un modelo in vitro (208).

La evidencia que apoya el potencial relevancia clínica de estos hallazgos ha sido obtenida de forma la detección de productos de desdoblamiento de la transferrina en broncoalveolar muestras de lavado de P. aeruginosa fibrosis quística con infección pacientes pero no en los de individuos normales (35).

Como los microorganismos requieren hierro para el crecimiento y la replicación, sus mecanismos de adquisición y almacenamiento de hierro se han desarrollado para satisfacer esas necesidades. bacterianos intracelulares de hierro es principalmente complejos con proteínas de almacenamiento como ferritina de hierro (229, 290). A adquirir el hierro del medio extracelular, aeróbica y bacterias anaerobias facultativas, así como hongos, sintetizar diversas de bajo peso molecular de Fe (III)-compactación ligandos colectivamente sideróforos llama (229). Estos compuestos poseen un gran afinidad por el hierro, que es probablemente importante en los sitios de infección (por ejemplo, la vía aérea), donde la disponibilidad de hierro para las bacterias es muy limitada debido a la competencia desde el host proteínas de unión a hierro. Como un ejemplo, para poder competir de hierro con eficacia, P. aeruginosa sintetiza y secreta dos tipos de sideróforos: pyochelin y pyoverdin (74, 75).

Estudios de nuestro laboratorio sugieren que puede desempeñar un pyochelin papel importante no sólo en la adquisición de hierro, sino también en P. aeruginosa lesión asociada tejido inflamatorio (38). El medio ambiente en los sitios de las infecciones por P. aeruginosa está repleta de ZO2 2 y H2O2 generado por los fagocitos y locales a través del redox acción de piocianina en las células diana (véase más adelante). Ferripyochelin puede actuar como un quelato de hierro catalíticamente activo en la formación de zoH (37) y puede mejorar oxidante mediada en endoteliales in vitro porcina arteria pulmonar (38) y epiteliales (36) la lesión celular. Por lo tanto, aunque la producción de sideróforos por P. aeruginosa es un mecanismo adaptativo para la obtención de hierro necesario bajo condiciones de estrés, lo mismo compuestos también pueden potenciar la lesión tisular mediada oxidante a través de la catálisis de zoH. Un papel similar para pyoverdin no ha se ha encontrado (69). Sin embargo, es posible que sideróforos producidas por otros microorganismos juegan un papel similar, pero hay Actualmente no hay datos disponibles para respaldar el presente. Otras fuentes potenciales de hierro catalíticamente activos relacionados a las interacciones huésped-microbio en vivo incluyen hierro liberado de la hemoglobina por la acción de la toxina bacteriana hemolisina, acogida a la reducción de la exposición de células derivadas de hierro bacterias compuestos tales como piocianina (discutido en una sección posterior), y / o la liberación de hierro intracelular de daños células de mamíferos o bacterianas en el microambiente. Independientemente de su origen, es necesario que extracelularmente catalizadores de hierro generados se encuentren próximas a la celda en Para facilitar lesiones zoH mediada, dada la limitada capacidad de difusión de zoH (116). Oxidantes mieloperoxidasa Derivados Coincidiendo con su producción de ZO2 2 y H2O2, estimulado fagocitos liberación humana uno de los dos peroxidasas distintas de sus gránulos. En el caso de los neutrófilos y monocitos, esta enzima es la mieloperoxidasa (MPO), mientras que de eosinófilos es peroxidasa de eosinófilos (OEP) (148). El interacción de la MPO y la OEP con formas H2O2 hypohalous ácidos (HOX, donde X 5 haluro). En general se cree que macrófagos o bien la falta de enzimas (169), sin embargo, datos recientes sugieren que esto puede no ser una verdad universal (79). Mieloperoxidasa es una glicoproteína (peso molecular, 150) que consiste en un par de pesados glucosilada (a)-luz (b) protómeros, cada uno de que contiene un átomo de hierro (225). La EPO es una glucoproteína ab, estructura similar a la hemi-MPO (148). Estas enzimas son catiónico, lo que les permite adherirse a las superficies celulares y tal vez aumentar su potencial de daño celular, aumentando la concentración local de ácido hypohalous en la membrana celular de destino (184, 212, 251, 277, 325). ácidos Hypohalous son oxidantes potentes que se conocen para tener varias efectos citotóxicos sobre las células de mamíferos y bacterias. La membrana de la célula integridad puede ser violado por la peroxidación de membrana y la oxidación y / o 2 descarboxilación de proteínas de membrana (, 322). Del mismo modo, la oxidación de los componentes de las bacterias respiratorias y la interferencia con la cadena de ADN bacteriano a la membrana interacción necesaria para la división bacteriana normal puede interrumpir el metabolismo celular y la replicación (249, 265). Neutrófilos activados y los monocitos pueden también generar cloraminas citotóxicos, tirosilo radical, y zoH través de una vía dependiente de la MPO (140, 148, 314). El óxido nítrico El óxido nítrico es citostáticos o citotóxicos tanto procariotas y las células eucariotas (105, 216). El principal mecanismo de lesión involucra la interacción de Noz con el hierro que contienen grupos funcionales de las enzimas clave del ciclo respiratorio (por ejemplo, la gliceraldehído- la síntesis de 3-fosfato deshidrogenasa) y con el ADN VOL. 10, 1997 PAPEL DE oxidantes en FISIOPATOLOGÍA MICROBIANA 3 Descargar de cmr.ASM.ORG - 20 de julio 2010 que conduce a la mutagénesis en las células diana (216). El óxido nítrico también pueden reaccionar con otras biomoléculas para formar nuevos compuestos que también son capaces de toxicidad. Por ejemplo, la formación de nitrosothiol grupos de proteínas puede conducir a la inactivación de enzimas o los cambios en función de las proteínas (216, 261). Estos grupos pueden reaccionar más a los grupos sulfhidrilo de la reticulación y por lo tanto iniciar una reacción en cadena (261). Además, Noz y sus derivados pueden formar agentes alquilantes tóxicos al reaccionar con aminas secundarias (156). Como se muestra en la Tabla 1, ONOO2 se genera por la reacción de ZO2 2 con Noz (19). Su capacidad de oxidar directamente sulfhidrilo los grupos y las bases de ADN, catalizar la membrana de hierro independiente peroxidación lipídica, y reaccionan con metales o metaloproteínas (Por ejemplo, la superóxido dismutasa [SOD]) para formar el nitronio tóxicos ion (NO22) ha llevado a algunos investigadores a sugerir que ONOO2 juega un papel más importante que su precursor en la mediación de Noz citotoxicidad (19, 144, 155, 247, 248, 287). Además, la evidencia sugiere que la protonación previa, pueden someterse a ONOO2 ruptura homolítica para formar zoH por un mecanismo independiente de hierro (19), sin embargo, la relevancia biológica de esta reacción ha no se ha abordado definitivamente. FUENTES de oxidantes encontrados por Microbios en VIVO Fuentes Endógeno Al igual que las células eucariotas, los microorganismos aeróbicos son continuamente expuestos a fuentes endógenas de especies de oxígeno tóxicos como una consecuencia del metabolismo aeróbico (12). Como se mencionó anteriormente, esto ocurre por la reducción secuencial de electrones univalente de las pruebas moleculares O2 para generar especies como ZO2 2, H2O2 y zoH.

Bajo ciertas condiciones, ruptura homolítica de H2O2 puede también producen zoH. Estas especies de oxígeno tóxicos también se pueden generar como subproductos de reacciones que involucran la glucosa oxidasa, la xantina oxidasa, y los grupos tiol y flavinas (73, 112, 252, 253). Por otra parte, la exposición a microbios a la UV o la irradiación g induce ZO2 2 de producción (200). organismos anaerobios son particularmente sensibles a los oxidantes derivados a través de los mecanismos anteriormente, ya que a menudo carecen de los mecanismos de defensa antioxidante observados en los organismos aeróbicos (véase más adelante). Un número de microorganismos, incluyendo Enterococcus faecalis (308), E. coli (150), Lactobacillus spp. (316), Streptococcus pneumoniae (316), y un número de Mycoplasma spp. (192, 204), también generan extracelular ZO2 2 y H2O2. Adicional estudios han demostrado que estos oxidantes pueden ejercer una serie de efectos beneficiosos y tóxicos tanto en el host y otros microorganismos. Por ejemplo, Lactobacillus productoras de H2O2 spp. inhiben la Neisseria gonorrhoeae y virus de inmunodeficiencia humana (VIH) de replicación in vitro (167, 332), lo que sugiere una inespecíficos mecanismo de defensa a los antibióticos derivados de la presencia de lactobacilos en la flora vaginal normal. Del mismo modo, en las mujeres con vaginosis bacteriana, los lactobacilos productores de H2O2 están notablemente ausentes de la flora vaginal (89). Por el contrario, ZO2 2 realizados por Mycoplasma pneumoniae puede inactivar la catalasa de la célula huésped, resultando en un daño progresivo oxidativo a las células infectadas in vitro (3). H2O2 S. pneumoniae derivados pueden desempeñar un papel en acogida daño celular en la neumonía neumocócica, como lo ha sido demostrado ser tóxico para las células epiteliales alveolares de rata en un in vitro modelo (86). La formación de placa dental y la posterior desarrollo de la gingivitis y la periodontitis también están relacionados en el equilibrio de H2O2 productores y degradantes H2O2- organismos en el microambiente oral (269). Los microorganismos también son expuestos continuamente a endógenamente Noz producidos por la desnitrificación (334). Este proceso es un modo distintivo de la respiración que es esencial para muchas formas de bacterias y hongos, pues implica la transformación de oxianiones de nitrógeno a N2, principalmente en condiciones de la tensión de oxígeno reducida o anaerobiosis estricta. La desnitrificación es controlado por un número de metaloenzimas, de los cuales el nitrito reductasa se ha identificado como la enzima responsable de la la conversión de nitritos a Noz (334). Dos mutuamente excluyentes nitrito reductasas se han identificado entre las bacterias desnitrificantes: un citocromo tetraheme cd situado en el periplasma de organismos gram-negativas, y una proteína vinculada con Cu- a la membrana citoplasmática de los organismos gram-positivos (334). Las ubicaciones de estas enzimas podrían limitar el potencial toxicidad de la producción endógena Noz, como posteriormente se Noz reducido rápidamente por una membrana citoplasmática asociada Noz reductasa.

Los datos recientes también demuestran la existencia de un sistema de NOS, en Nocardia spp. (64), la primera confirmación de este sistema en los microorganismos. La estequiometría de los productos , hace referencia a los sustratos utilizados, los requisitos de cofactor, y la inhibición por NG-nitro-L-arginina se consideraron similares a los observados en mamíferos NOS (64). Hay evidencia de que los eritrocitos infectados con Plasmodium falciparum también puede generar a través de Noz NOS ya producen una soluble factor que es capaz de evocar la producción Noz en los tejidos del huésped (113). Informes de otras NOS microbiana es probable que aparezcan en el futuro. Las fuentes exógenas oxidantes derivados de los fagocitos y su papel en la defensa del huésped. La principal fuente de estrés oxidativo exógeno para los patógenos bacterias durante el proceso de infección activa es su ataque de acogida fagocíticas células. Los fagocitos utilizar los citotóxicos efectos de muchos de los oxidantes descritos anteriormente como un componente de su mecanismo de defensa del huésped (Fig. 1). Cuando un fagocito encuentra un microorganismo, éste está rodeado por un porción de la membrana del fagocito, que luego se invagina, formando un fagosoma discretos (148). Este proceso conduce a mayor consumo de oxígeno e inicia un fagocito complejos bioquímica del sistema de señalización que activa una única asociada a membrana complejo oxidasa NADPH-dependiente (67). Esta enzima reduce univalently O2 ZO2 2, que luego se secretada en el fagosoma (67). Allí, ZO2 2 a dismutes H2O2. Estos compuestos tóxicos también pueden tener fugas extracelularmente el fagosoma se está cerrando.

Tras la fagocitosis, los microorganismos son sometidos a FIG. 1. Listado de los procesos que conducen a intraphagosomal oxidante mediada asesinatos microbiana. CA, las cloraminas. Tenga en cuenta que la producción de óxido nítrico se produce sólo en los fagocitos con un óxido nítrico sintasa inducible. Reproducido de referencia 209 con permiso del editor. 4 MILLER Y CLIN BRITIGAN. Microbiol. REV. Descargar de cmr.ASM.ORG - 20 de julio 2010 insulto como principal fagocito (o azurófilos) citoplasmática gránulos se fusionan con el fagosoma. Además de la MPO, estos gránulos contienen principalmente hidrolasas (hidrolasas ácidas, lisozima, proteasas neutras, desoxirribonucleasas, etc), que son probablemente responsable de la descomposición de organismos muertos (31). Secundaria (o específica) se fusionan con los gránulos citoplasmáticos membrana plasmática externa antes de los gránulos primarios hacer, lo que secretan su contenido (lactoferrina, lisozima y vitamina B12-La unión a proteínas) extracelular (283). Las membranas de estos gránulos secundarios también contienen una serie de funcionalmente las proteínas importantes, incluyendo CD11b/CD18, el receptor formil-metionil-leucil-fenilalanina, y el citocromo b558 (48). La fusión de estos gránulos con el plasma membrana sirve para reforzar o mantener varias respuestas celulares (29). La importancia del sistema NADPH-oxidasa de acogida actividad microbicida se ejemplifica en individuos con enferme
ades crónicas enfermedad granulomatosa (CGD), un grupo de trastornos hereditarios que son cada una caracterizada por defectos en la NADPH-oxidasa complejo que resulta en una falta de fagocito ZO2 2 Producción (299). La NADPH-oxidasa requiere el montaje de su membrana y componentes para la generación de citosólica de las vías respiratorias estallar.

Del mismo modo, los defectos genéticos en esta enzima observada entre los pacientes con enfermedad granulomatosa crónica se caracterizan por su localización de la membrana o citosol. Aproximadamente el 60% de los pacientes tienen un defecto EGC ligada al cromosoma X en la membrana b componente del citocromo como consecuencia de mutaciones en el gp91phox (55%) o p22phox (5%) del gen que codifica para grandes y subunidades pequeñas, respectivamente (236, 267, 300). Los pacientes con autosómica defectos más comunes la falta del componente citosólico, p47phox, y representan aproximadamente el 35% de los casos (55, 68). Menos del 5% de los pacientes carecen de la CGD p67phox citosólica componente (68). Con independencia de la localización del defecto, la manifestaciones clínicas de las diferentes formas genéticas de la EGC son bastante similares.

Estas personas sufren de recurrentes piógeno infecciones por microorganismos que normalmente son rápidamente asesinado por oxidantes: Staphylococcus aureus, bacilos gramnegativos entéricos, Aspergillus spp., y Candida spp. Las complicaciones infecciosas, , que puede afectar casi cualquier órgano o sistema, típicamente comienza en la la infancia y se repiten durante toda la infancia y la adolescencia. Aunque se asocian a estados de la neutropenia, infecciones con otros organismos patógenos, tales como P. aeruginosa son pocas veces se encuentra en pacientes CGD (299). Los datos in vitro han demostrado la capacidad de los neutrófilos para destruir P. aeruginosa (127, 145, 196, 221, 328). Este proceso es marcadamente mayor en presencia de suero que el organismo opsoniza con el complemento y la inmunoglobulina para promover más fagocitosis por los neutrófilos eficiente (328). Sin embargo, adicionales en las observaciones in vitro sugieren que los oxidantes no son críticos por matar a neutrófilos mediada por P. aeruginosa. Los neutrófilos de pacientes CGD tienen la misma capacidad que los neutrófilos normales para matar a P. aeruginosa (145). Sin la presencia de ambiente O2, los neutrófilos son incapaces de generar ZO2 2 y H2O2. Sin embargo, sus habilidades para matar a P. aeruginosa en aeróbica y condiciones anaeróbicas parecen ser similares (196). Esto está en A diferencia de resultados con S. aureus, en el que los neutrófilos son no pueden matar el organismo en condiciones anaeróbicas in vitro (197). Puede ser que ZO2 2-mecanismos independientes de neutrófilos matar, como las relacionadas con proteínas derivadas de gránulo and proteases, are more important in P. aeruginosa elimination (311). However, these findings do not eliminate the possibility that neutrophil-derived oxidants increase the effectiveness of the O2-independent killing mechanisms. Phagocyte-derived H2O2 may also be converted intra- or extracellularly to HOCl and the longer-lived chloramines in the presence of chloride and myeloperoxidase.

Por otra parte, MPO can catalyze the reaction of zO2 2 and HOCl to form zOH (232). All of these compounds are known to have a number of cytotoxic effects in vitro (313). However, their overall significance in in vivo microbicidal activity is unclear, as patients with MPO deficiency demonstrate delayed killing of fungi and bacteria but are normally resistant to most infections (237). Of all patients recognized with this disorder (a prevalence of approximately 1 in 2,000 of the general population) (225), only a few have had serious infections (58, 166). The majority of these patients had visceral or disseminated candidiasis (58, 179). Three of these patients had concomitant diabetes mellitus (58, 179), perhaps indicating that the clinical morbidity associated with MPO deficiency requires an additional defect in host defense.

Phagocytes may also participate in mode 1 and 2 bacterial killing by generating H2O2 as described by Imlay et al. (149, 153). As discussed above, the interaction of exogenous H2O2 at low concentrations with intracellular Fe21 in E. coli results in DNA damage mediated by the ferryl radical. Bacterial exposure to higher H2O2 concentrations resulted in killing by a separate oxidative mechanism. Perhaps a more physiologically significant mechanism involved in phagocyte-mediated oxidant generation and microbial toxicity involves the iron-catalyzed intra- or extracellular reaction of zO2 2 and H2O2 to form zOH. Aunque hay una limited amount of free iron available for this reaction to take place in vivo, multiple potential host and microbial catalytic iron complexes exist, as discussed previously. In vitro studies have demonstrated that increased bacterial iron concentrations enhance zOH-mediated killing of S. aureus by H2O2, human monocytes, and neutrophil-derived cytoplasts (142, 256). However, the role of zOH-mediated killing of S. aureus by intact human neutrophils remains unresolved (70, 257). En addition, killing of Leishmania donovani chagasi promastigotes by H2O2 appears to involve iron-dependent zOH formation (329), but these studies have not yet been extended to phagocyte sistemas. The role of iron in microbe-phagocyte interactions is clearly complicated, since Byrd and Horwitz have shown that conditions that modulate phagocyte iron concentration appear to affect intracellular microbicidal activity against Legionella pneumophila and M. tuberculosis in opposite ways (46, 47). Recently, NOz has been increasingly recognized as another phagocyte-derived oxidant involved in microbicidal activity. Su synthesis requires a NOz synthase, of which there exist constitutive and inducible isoforms (see above) (216).

The inducible enzyme has been definitively demonstrated in murine phagocytic cells and can be induced by a number of cytokines and lipopolysaccharides (216, 295). Despite efforts by many investigators, however, the ability to detect NOz production by human mononuclear phagocytes has been modest at best under conditions where NOz production by murine phagocytes is readily apparent (49, 117, 159, 160, 223). Recent data have demonstrated that human mononuclear phagocytes can produce constitutive NOz synthase (255, 312). The inducible NOz synthase mRNA and protein are generated in response to lipopolysaccharide and/or gamma interferon stimulation, demonstrating that human phagocytes appear to possess the necessary "machinery" to synthesize NOz. More direct evidence for NOz production by human macrophages has been demonstrated by the recent findings of Nicholson et al. (227). Una average of 65% of alveolar macrophages in bronchoalveolar lavage specimens from 11 patients with untreated, culturepositive pulmonary tuberculosis contained NOz synthase mRNA and functional NOz synthase expression. Of note, only 10% of alveolar macrophages from normal subjects demon- VOL. 10, 1997 ROLE OF OXIDANTS IN MICROBIAL PATHOPHYSIOLOGY 5 DOWNLOADED FROM cmr.ASM.ORG - July 20, 2010 strated similar findings. However, despite these reports, the quantity of NOz generated under a number of conditions was very small (312). In addition, studies where NOz production is equated with nitrite production may falsely overestimate the true quantity of NOz synthesis, as shown by Klebanoff and Nathan, who demonstrated that human neutrophils can synthesize nitrites via the catalase-catalyzed conversion of azide to nitrite in the presence of phagocyte-generated H2O2 in vitro (168). The primary microbicidal effect of phagocyte-derived NOz appears to involve intracellular pathogens. A clear role in pyogenic bacterial infections has not been demonstrated. Por treating murine-activated macrophages in vitro with NG-monomethyl- L-arginine, a competitive inhibitor of nitrate and nitrite synthesis from L-arginine, a number of investigators have implicated NOz as having microbiostatic and/or microbicidal activity against pathogens such as Cryptococcus neoformans (117), Toxoplasma gondii (1), Mycobacterium bovis (100), Leishmania major (180, 182), Schistosoma mansoni (160), and others (223, 261). Further studies involving an in vivo model of murine leishmaniasis have demonstrated that NOz plays an important role in containing the extent of infection and decreasing the overall organism load (92, 181). A similar result was observed by Boockvar et al. in an in vivo model of murine listeriosis (28). Although the importance of NOz production to murine macrophage function is now well established, these data are not directly applicable to human phagocytes, because the existence of a role for NOz in human phagocyte microbicidal activity is less clear. Recent data by Vouldoukis et al. suggest that the killing of L. major by human macrophages is mediated by NOz, whose production is induced after cell activation via ligation of the low-affinity receptor for immunoglobulin E (FcERII/CD23 surface antigen) (307). This receptor is upregulated in cutaneous leishmaniasis. Additional in vitro data imply that tumor necrosis factor alpha (TNF-a) and granulocyte-macrophage colony-stimulating factor stimulate human macrophages to restrict the growth of virulent Mycobacterium avium by a mechanism involving NOz (80). The previously discussed findings by Nicholson et al. (227) also suggest that NOz may be an important component of host defense against pulmonary tuberculosis. Likewise, Bukrinsky et al. reported that lipopolysaccharide or TNF-a-activated HIV-infected monocytes exhibit enhanced NOz production (44). In support of these findings, the authors detected RNA encoding the inducible NOz synthase in postmortem brain tissue from an AIDS patient with advanced HIV encephalitis. Nitric oxide may also contribute to the killing of staphylococci by neutrophil cytoplasts (anucleate, granulepoor, motile cells) which rapidly took up and killed the bacteria by a mechanism inhibited by NG-monomethyl-L-arginine (194). In contrast to these various studies, a direct comparative study between murine and human macrophages revealed that activated murine but not human macrophages demonstrated enhanced NOz production and antimicrobial activity against Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani (223). Although not directly compared with murine macrophages, NOz production contributes minimally to human macrophage-mediated killing of Cryptococcus neoformans and Schistosoma mansoni (49, 158), organisms which are killed by a NOz-mediated mechanism in murine macrophages (117, 160). This suggests that NOz makes a minimal contribution to the overall microbicidal activity against these pathogens in the human host. Thus, there are increasing data supporting the concept that human phagocytes can produce NOz, albeit in small quantities relative to their murine counterparts. However, these data are somewhat difficult to interpret, as the frequency with which negative findings are reported by laboratories is often quite low. This capability to synthesize NOz appears to be mediated via the classic NOS pathway. The microbicidal activity of human, like murine, phagocyte-derived NOz, if and
when it is generated, could contribute to host protection against intracellular organisms. However, the contribution of NOz relative to the other phagocyte antimicrobial mechanisms known to be effective against these and other pathogens has yet to be established. Other oxidant sources and their contribution to microbicidal actividad. Although phagocytes are the primary source of microorganism exposure to oxidants in mammalian hosts, other mechanisms of oxidant production exist and probably contribute to microbial oxidant stress. As discussed in a previous section, microorganisms such as Nocardia and Lactobacillus spp. produce NOz and H2O2, respectively, which may in turn have toxic effects on other microorganisms in close proximity (64, 89, 167, 332). In addition, endothelial cells produce NOz, zO2 2, and H2O2 in response to a number of stimuli, including inflammation (216). Feng et al. have recently suggested that endothelial cell-derived NOz could protect these cells from infection with Rickettsia conorii (96). In an experimental model, pulmonary (tracheal and alveolar) epithelial cells also demonstrate luminal H2O2 production, which is enhanced after stimulation by phorbol myristate acetate and platelet-activating factor (165). Epithelial cells from cystic fibrosis patients have been shown to consume two- to threefold more oxygen than do normal cells, providing indirect evidence of a highly oxidative environment in a population known to have a chronically high organism load (296). These endothelial cell- or epithelial cell-derived compounds may exert microbial oxidant stress either alone or via their reaction by-products such as ONOO2 and/or zOH in the extracellular space. Además, these oxidants may interact with phagocyte-derived oxidants, cytokines, and other compounds to potentiate the microbial insult. Several antimicrobial agents used in the treatment of clinical infections, in addition to blocking key enzymes and other metabolic functions of microorganisms, produce reactive oxygen intermediates that are capable of damaging other biomolecules. For example, b-lactam antibiotics (penicillins and cephalosporins) have been shown to oxidatively damage DNA and deoxyribose in the presence of iron and copper salts, consistent with an zOH-mediated mechanism (246). In addition, the polyunsaturated structure of the polyene antifungal antibiotics (amphotericin, natamycin, and nystatin) gives them the propensity to oxidize to form peroxy radicals and thiobarbituric acid-reactive aldehyde fragments (281). These interactions can then lead to the generation of other oxygen-centered radicals capable of inciting further microbial injury. These newly recognized antibiotic effects may prove to be an important component of their biologic activities. Likewise, a number of compounds undergo rapid redox cycling under aerobic conditions, potentially resulting in an additional source of extracellular oxidants for microbial encounter (135, 191). These compounds are univalently reduced to free radicals by cellular systems. In the presence of O2, these reduced molecules are then reoxidized, with the resulting transfer of that electron to O2, hence forming zO2 2 and H2O2, the latter via zO2 2 dismutation. Examples of such compounds include pharmacologic agents such as adriamycin, bleomycin, and nitrofurantoin (191). The P. aeruginosa secretory product pyocyanin works by a similar mechanism. This compound is a phenazine-derived pigment that can undergo redox cycling to induce both intra- and 6 MILLER AND BRITIGAN CLIN. MICROBIOL. REV. DOWNLOADED FROM cmr.ASM.ORG - July 20, 2010 extracellular zO2 2 and H2O2 production from O2 in both eukaryotic and prokaryotic cells (134, 135, 218). This process contributes to cell death through the diversion of electron flow from normal biologic pathways into those leading to toxic oxidant generation. This pyocyanin-induced production of zO2 2 and H2O2 also can lead to the formation of zOH in the presence of a catalytic iron source (38, 208). Pyocyanin production increases under conditions of nutritional deprivation and oxidative stress (136). Interestingly, however, P. aeruginosa itself is relatively insensitive to pyocyanin and seemingly escapes oxidant-mediated injury during production of or exposure to this compound (136). This may be explained in part by its low endogenous levels of NADH/NADPH, its lack of NADPH:pyocyanin oxidoreductase, and/or its high levels of SOD and catalase. In addition to its redox capabilities, pyocyanin has numerous in vivo and in vitro effects which could play a role in the pathogenesis of clinical infections. For example, the addition of pyocyanin, at concentrations detectable in pulmonary secretions of individuals with P. aeruginosa infection, to human ciliated nasal epithelial cells (4, 317, 318) and sheep tracheal epithelial cells (157) results in a loss of ciliary function as well as a decrease in in vivo tracheal mucus velocity in the sheep model (222). The effect on sheep cilia could be negated by the simultaneous presence of catalase, suggesting an oxidant-mediated mechanism (222). This process may contribute to the difficulty that cystic fibrosis patients experience in mobilizing their secretions (222). Other effects of pyocyanin potentially relevant to microbial killing and inflammatory tissue injury include those on stimulated neutrophils to alter zO2 2 production and degranulation, host cell NOz production, and lymphocyte proliferation and differentiation (207, 219, 220, 230, 282, 305, 310). MECHANISMS OF MICROBIAL DEFENSE AGAINST OXIDANTS Avoidance of Encounters with Phagocyte-Derived Oxidants As microbial killing by phagocytes is a multistep process, microorganisms have likewise developed a sequential series of defense strategies to counteract this process. Some microorganisms secrete toxins to kill the phagocyte before they can be killed by it. Examples include the production of streptolysin by Streptococcus spp. (22), leukocidin by Staphylococcus aureus (263), and the Clostridium septicum toxin (193). Other organisms resist phagocytic uptake by covering their surfaces with hydrophobic capsules (Neisseria meningitidis, pneumococci) (103, 141, 147, 158, 258-260, 324). Pathogenic mucoid strains of P. aeruginosa synthesize alginate, an exopolysaccharide. En addition to aiding in avoiding phagocytic uptake, alginate has the ability to scavenge reactive oxygen intermediates, suppress leukocyte function, and promote bacterial adhesion (11, 82, 87, 177, 239, 280). This may be of particular clinical relevance, as airway isolates from individuals with cystic fibrosis commonly demonstrate alginate production (82, 239). P. aeruginosa also requires a unique glucose-dependent pathway for phagocytosis by macrophages (13). This may enhance its pathogenicity in the bronchoalveolar space, where concentrations of glucose and other carbohydrate are low. Acidification within the phagocytic vacuole is an important process to maximize the spontaneous dismutation of zO2 2, hydrolase activity, and phagosome- lysosome fusions. Inhibition of this acidification process has been described for Legionella pneumophila (146) and Toxoplasma gondii (279). Although phagocyte-derived oxidants are important mediators in microbial killing, some organisms can survive the encounter to then inhibit phagosome-lysosome fusion and avoid enzymatic attack by hydrolytic enzymes. Esta process is poorly understood but has been demonstrated among some mycobacteria (143), T. gondii (143), Chlamydia spp. (143), and others (41, 146). Other organisms, such as Listeria monocytogenes (60), Shigella flexneri (274), and Trypanosoma cruzi (5), are able to escape from the phagosome by the secretion of membrane-damaging cytolysins. Another key virulence factor allowing for the avoidance of host defense mechanisms has been identified in a number of Yersinia spp. In Yersinia enterocolitica, a 51-kDa periplasmic protein encoded by the yop-51 gene shares amino acid sequence identity with the catalytic domain of several protein tyrosine phosphatases (PTPases) (120). Activation of protein tyrosine kinases is an important signaling mechanism in many cells, including macrophages. By interfering with host signaling pathways, Yersinia spp. have the potential to modify the host immune response, which probably explains the importance of this process as a virulence factor. The yop-51 gene resides on a naturally occurring 70-kb plasmid, and its mutation alters the virulence of the organism (26). An analogous gene, yopH, encodes a similar PTPase in Y. pseudotuberculosis. Además work has characterized the crystalline structure and active site of these proteins (294, 331). The specific gene and corresponding PTPase have not been determined for Y. pestis, but preliminary studies reveal that the yop-51/yopH gene is highly conserved in this organism (242). Defense Strategies Specific for Oxidants Nonenzymatic. Exposure to intraphagosomal oxidants is a fatal event for many microorganisms. However, some organisms have evolved an ability to inhibit the NADPH- oxidasedependent oxidative burst and thus to inhibit reactive oxidant production within the phagosome (Fig. 2). This appears to be particularly important for intracellular pathogens as it aids in survival within the phagosome. For example, the lipophosphoglycan present on the membrane of Leishmania major and L. donovani (analogous to lipopolysaccharide in bacteria) inhibits protein kinase C activity in macrophages (30, 104), resulting in suppression of the respiratory burst and ultimately of zO2 2 de producción. This inhibition of macrophage protein kinase C activity also impedes macrophage chemotactic locomotion and interleukin-1 (IL-1) production. Legionella pneumophila se- FIG. 2. Overview of bacterial defense mechanisms against oxidative killing inside the phagosome. VOL. 10, 1997 ROLE OF OXIDANTS IN MICROBIAL PATHOPHYSIOLOGY 7 DOWNLOADED FROM cmr.ASM.ORG - July 20, 2010 cretes a compound shown to inhibit the neutrophil oxidative burst (272). Leishmania donovani (114) and Legionella micdadei (271) produce extracellular acidic phosphatases that block zO2 2 formation in vitro. The mechanisms of these effects have not been further elucidated, however. Antioxidant scavengers unique to specific pathogens have also evolved to protect microorganisms from phagocyte-derived oxidants. As noted above, P. aeruginosa produces alginate, an exopolysaccharide capable of scavenging oxidants (177, 280). In addition, the phenolic glycolipid of mycobacteria and the lipophosphoglycan of L. donovani are effective in scavenging zOH and zO2 2; these characteristics may enhance the intracellular survival of the organisms (61, 226). Cryptococcus neoformans is known to produce large amounts of mannitol both in vivo and in vitro (323). Mannitol in high concentrations has the ability to scavenge reactive oxygen species. Thus, its production by C. neoformans may be a protective mechanism by which the organism protects itself from oxidative killing by host phagocytes. Chaturvedi et al. have recently demonstrated, by using a low-mannitol-producing mutant of C. neoformans, that the ability of C. neoformans to produce and accumulate mannitol may influence its tolerance to heat and osmotic stresses and its pathogenicity in mice (62) through the scavenging <
r>of reactive oxygen intermediates (63). The formation of heat shock proteins (HSP) by ingested bacteria may represent another adaptive mechanism. HSP production can be induced by increased temperature and/or oxidant exposure as a means of protection against both heat and oxidant damage. In Mycobacterium tuberculosis and Mycobacterium leprae, a strongly immunogenic antigen can be recognized by use of monoclonal antibodies (327). Production of this protein can be induced by stress, which may include phagocytosis. Severe stresses also increase the production of antioxidant enzymes such as SOD. There exists some evidence that HSP may also play a role in the regulation of antioxidant enzyme production in E. coli (293); this is discussed in more detalle a continuación. Little is known about microbial defense against NOz. During the process of denitrification, microorganisms appear to limit toxicity by keeping endogenous NOz levels very low (334). In an in vitro model, extracellularly generated NOz was inactivated by the P. aeruginosa-derived phenazine pigment pyocyanin (310). Once phagocytized, microorganisms may have evolved a strategy to inhibit host nitric oxide synthase analogous to what has evolved for the NADPH-oxidase complex. However, at present, this has not been reported. Enzymatic. Microorganisms have developed highly specific and effective enzymatic pathways of oxidant inactivation, including those catalyzed by SOD, catalase/peroxidase, and glutathione (GSH) in combination with glutathione peroxidase and glutathione reductase (122, 137). (See Fig. 2 and Table 1 for chemical reactions.) Glutathione serves as a substrate for the H2O2-removing enzyme glutathione peroxidase. It can then be redox cycled via glutathione reductase for further H2O2 removal. GSH is also an zOH scavenger. Eukaryotic cells depleted of GSH exhibit increased susceptibility to oxidant-mediated killing (205). There are also data suggesting that GSH depletion is involved in HIV replication (162, 286). The importance of this antioxidant system in prokaryotes, however, has not been clearly established. GSH reductase-negative E. coli mutants do not demonstrate an increased susceptibility to H2O2-mediated stress compared with the isogenic parental strain (118). Sin embargo, there are data suggesting that GSH may facilitate the deactivation of E. coli aconitase and other [4Fe-4S]-containing dehydratases that have been oxidatively inactivated by zO2 2 (111). Proteins immunologically related to GSH have been demonstrated in other bacterial species and in other strains of E. coli (240). Recently, Moore and Sparling have identified a GSH peroxidase homolog gene, gpxA, in Neisseria meningitidis. The amino acid sequence of this gene is highly homologous to GSH peroxidases found in other bacterial species (217). Por lo tanto, there may be several types of GSH-metabolizing proteins in bacteria which serve a similar purpose, and their distribution may even vary within a single species. Protozoa such as trypanosomes and leishmaniae produce trypanothione (93). Lo may have an analogous function to GSH in that it functions to maintain thiol redox within the organism and as a defense mechanism against oxidants, xenobiotics, and heavy metals. The importance of trypanothione to parasite survival can be exemplified by organism exposure to D,L-a-difluoromethylornithine, an antiparasitic agent used in the treatment of human African trypanosomiasis. D,L-a-Difluoromethylornithine inhibits parasite ornithine decarboxylase, which results in decreased cellular trypanothione levels among other effects (93). Why these organisms have evolved to produce trypanothione in addition to GSH is unclear. Considerably more data are available on the distribution, structure, and regulation of microbial catalases and peroxidases (186). The antioxidant action of these enzymes is to catalytically convert H2O2 to H2O and O2. Nearly all aerobic and facultatively anaerobic microorganisms, with the exception of the Streptococcus spp., synthesize at least one form of catalase and/or peroxidase (201). The majority of obligate anaerobes lack this capability (201). These proteins are characterized by structural diversity between different organisms and even within the same organism. The most common form consists of a homotetramer with one protoheme IX per subunit. Most bacteria produce two catalases, whereas others such as Klebsiella pneumoniae and P. aeruginosa have the ability to produce multiple catalases under specific growth conditions (115, 136). The two structurally distinct catalases of E. coli, termed hydroperoxidase I (HPI) and hydroperoxidase II (HPII), have been the most extensively studied (65, 66). HPI, a bifunctional catalase-peroxidase encoded by katG, contains two protoheme IX groups associated with a tetramer of identical 80-kDa subunits and is localized in the periplasmic space. HPII, a monofunctional catalase encoded by katE, consists of six heme d isomers associated with a hexameric structure of 84.2-kDa subunits and is found solely in the cytoplasm. The relative levels of HPI and HPII are controlled by two different regulons that respond to different environmental stimuli (186). HPI is synthesized preferentially in response to oxidative stress (H2O2), whereas HPII is produced in response to nutrient depletion as occurs in the stationary growth phase (188). Por lo tanto, not only are HPI and HPII different structurally and genetically, but also the processes controlling their synthesis respond to different stimuli and involve different mechanisms. The two catalases of Bacillus subtilis have been studied in comparison and appear to show some resemblance to E. coli HPI and HPII with regard to their structure and mechanism of control (185, 187). Among other bacterial species, the catalases of several other members of the Enterobacteriaceae family exhibit homology to E. coli HPI and HPII (298). The complexity of bacterial catalase expression and regulation can be demonstrated by the reported correlation between the loss of catalase production and isoniazid resistance among Mycobacterium tuberculosis isolates (98, 330). Diferente amounts of catalase production have been found in a number of organisms in response to nutrient depletion and in association with their susceptibility to phagocyte killing (136, 188, 8 MILLER AND BRITIGAN CLIN. MICROBIOL. REV. DOWNLOADED FROM cmr.ASM.ORG - July 20, 2010 197). For example, the growth of P. aeruginosa in limitedsuccinate media resulted in increased catalase activity and the appearance of additional catalase isoforms compared to the catalase activity in the same organisms grown under nutritionally replete conditions (136, 210). Mandell demonstrated that neutrophils easily killed low- but not high-catalase-producing Staphylococcus aureus strains (197). This difference correlated with in vivo lethality in a mouse model. Likewise, catalasedeficient E. coli mutants exhibit an increased susceptibility to phagocyte-mediated killing (121). Another mechanism of oxidant inactivation used by microorganisms involves SOD. The production of this group of enzymes is a key defense strategy aimed at the elimination of zO2 2. Not only does this decrease the possibility of direct zO2 2-mediated toxicity, but also it prevents zO2 2-mediated reduction of iron and subsequent zOH generation via the Haber- Weiss reaction. There are three common forms of SOD found in nature (12). Eukaryotes and some higher fungi predominantly produce CuZnSODs, homodimers (molecular weight 32,000) with two noncovalently linked identical subunits containing one atom each of copper and zinc. A few species of bacteria have also been found to contain CuZnSOD; some of these include Stenotrophomonas (Pseudomonas) maltophila (289), Brucella abortus (18), several Haemophilus spp. (171, 172, 176), E. coli (21), N. meningitidis (173), L. pneumophila (291), and Salmonella spp. (52). All bacteria, including obligate anaerobes, produce either FeSOD, MnSOD, or both. Como CuZnSOD, these enzymes exist as subunits (molecular weight 23,000) linked as dimers in FeSOD and dimers and tetramers in MnSOD. The metal content of both isozymes varies between 1 and 2 atoms per dimer. Most SODs are cytoplasmically located, although a few are located on or secreted through the cytoplasmic membrane (16, 21, 270). In general, FeSOD predominates in anaerobic organisms whereas MnSOD is more commonly found among aerobic organisms. Although variations in the FeSOD content have been observed in bacteria producing both isoenzymes, it is the control of MnSOD that is usually responsible for modulating the total level of SOD in bacteria. Like catalase, microbial SOD regulation and genetics have been most extensively studied in E. coli (302), where SOD expression is dependent on a number of environmental stimuli. FeSOD, encoded by the sodB gene, is produced constitutively in E. coli grown aerobically or anaerobically but is upregulated when grown anaerobically in the presence of nitrate. MnSOD becomes the predominant form when the cell is exposed to oxidative stress. In addition, a hybrid form containing Fe and Mn has also been isolated in vitro (85). It appears that, functionally, FeSOD provides E. coli with the first line of defense against zO2 2 and MnSOD is subsequently recruited in circumstances of increased oxidative stress. MnSOD gene (sodA) expression is governed by a number of regulon proteins, such as the Fur proteins and those under the control of the sox gene locus, including the Arc protein (119, 228, 304). These proteins are made in response to such stimuli as iron availability and oxygen/oxidant exposure, respectively. Interestingly, the Arc regulatory protein is also involved in the control of aconitase synthesis, suggesting that increased MnSOD is necessary to protect increased cellular concentrations of aconitase (109). Evidence suggests that MnSOD regulation also occurs at the posttranscriptional and posttranslational levels (244). In recent work, Hassett et al. have begun to characterize a similar system in P. aeruginosa, which, like E. coli, possesses both an iron- and manganese-cofactored SOD (136, 138). Notably, P. aeruginosa has approximately four to five times the SOD activity reported for E. coli (136). When cloned, the genes encoding the MnSOD (sodA) and FeSOD (sodB) revealed a 50 and 67% sequence homology with the respective E. coli SODs. The relative quantities of the FeSOD or MnSOD isoenzyme produced appear, as in E. coli, to be dependent on nutritional availability and the degree of oxidative stress (136). Some pathogens have evolved the ability to localize SOD activity to their extracellular environment as a means of resisting oxidant attack. For example, Mycobacterium tuberculosis secretes an FeSOD, whereas Legionella pneumophila and E. coli possess a periplasmic CuZnSOD (21, 270). Nocardia asteroides has a unique SOD associated with the outer cell wall, which can be selectively secreted extracellularly. This SOD differs significantly from those isolated from other bacteria in that it contains equimolar amounts of Fe, Mn, and Zn (16). Although N. asteroides induces an oxidative burst in human phagocytes, it is not readily killed by this mechanism. Posterior in vitro and in vivo studies have demonstrated that this resistance to phagocyte-mediated killing is dependent on the production and secr
tion of SOD by the organism (15, 17). Initial killing and/or enhanced clearance of N. asteroides was observed in organs obtained from infected mice given a monoclonal anti-SOD antibody-treated N. asteroides. This effect was not observed in mice given a nonspecific nocardial antibody. Thus, the extracellular localization of bacterial SOD may be an important determinant in the pathogenesis of infection for N. asteroides and other pathogens. The importance of microbial SOD production can be appreciated when studying SOD-deficient organisms. MnSOD- and FeSOD-negative mutants have been obtained from E. coli (303). However, with the recent discovery of the periplasmic E. coli CuZnSOD (21), studies with these mutants warrant qualification. Nonetheless, these organisms exhibit extreme sensitivity to oxidizing agents such as paraquat and methylene blue and are more susceptible to phagocyte-mediated killing (53, 121). Fe- and Mn-SOD-deficient double mutants demonstrate a marked increase in oxygen-dependent mutagenesis (94). Amino acid biosynthesis and membrane integrity also appear to be affected (151, 152). However, in vitro data obtained with E. coli suggest that overexpression of SOD may also be deleterious by accelerating H2O2 production in the organism upon its exposure to oxidative stress (276). ROLE OF OXIDANTS IN VIRAL INFECTIONS There is recent evidence that oxidants, whether derived from phagocytes or other sources, play a role in the pathogenesis of viral infections. The majority of work has centered around HIV. HIV infection is associated with a proinflammatory state in the host, resulting in high levels of circulating cytokines, including TNF-a, IL-1a, IL-1b, IL-2, IL-6, alpha interferon (IFN-a), and IFN-g (262). Although it has been shown that some of these cytokines can activate HIV replication in the infected host cell directly (241), cytokine activation of phagocytes and other cells can also stimulate oxidant production. Oxidants also have direct effects on HIV replication. Legrand- Poels et al. demonstrated that the addition of exogenous H2O2 to a latently HIV-infected T-cell line (U1) resulted in increased replication of the HIV-1 provirus (178). Schreck et al. confirmed these findings in Jurkat T cells and provided further insight into the mechanism of activation (275). Este proceso, like direct cytokine activation of HIV, is mediated by the induction of NF-kB, a ubiquitous transcription factor that is recognized by the HIV promoter (275). Likewise, Sandstrom et al. showed that HIV gene expression enhanced T-cell susceptibility to H2O2-induced apoptosis (273). HIV-infected cells may be uniquely sensitive to oxidant VOL. 10, 1997 ROLE OF OXIDANTS IN MICROBIAL PATHOPHYSIOLOGY 9 DOWNLOADED FROM cmr.ASM.ORG - July 20, 2010 stress, as a number of studies have shown them to exhibit low levels of GSH, the main intracellular defense against oxidants. HIV-infected patients demonstrate decreased GSH levels in blood and peripheral blood mononuclear cells relative to those in normal patients, and this decrease becomes more pronounced with advanced disease (81). More specifically, Staal et al. found that in patients with symptomatic AIDS, GSH concentrations in CD8 and CD4 T cells are 62 and 63%, respectively, of those found in seronegative controls (286). El más grande decreases in GSH levels were seen in those patients with advanced infection. Not only does this decrease in intracellular GSH levels leave the infected cell susceptible to the direct effects of oxidants, but also it leads to increased NF-kB expression, resulting in further activation of HIV replication (285). Using a HeLa cell line transfected with the tat gene from HIV-1, Flores et al. found that the expression of the regulatory Tat protein, essential for virus replication, suppresses the expression of cellular MnSOD (102). These cells also exhibited other evidence of increased oxidative stress manifested by elevated levels of carbonyl proteins and decreased cellular sulfhydryl content (102). Thus, HIV-mediated modification of host antioxidant enzymes may be an important component in mediating ongoing HIV infection and the ultimate progression to severe immunodeficiency. This process may be further altered in the presence of opportunistic pathogens. These advances in the understanding of the pathogenesis of HIV infection have prompted investigations into the use of antioxidants as therapy for HIV-infected individuals. In vitro studies with an HIV-infected human promonocytic cell line have demonstrated that HIV expression can be decreased by treatment of the cells with GSH, glutathione ester, or N-acetylcysteine (162, 195). Each of these compounds increases intracellular thiol concentrations and, as a result, inhibits NF-kB and ultimately HIV expression. These observations have led to studies of HIV-infected patients to determine whether the administration of N-acetylcysteine or L-2-oxothiazolidine-4- carboxylic acid (Procysteine) may alter disease progression (81, 161). Although both of these compounds were found to increase intracellular GSH levels in treated patients, there were no significant differences in CD4 cell counts, viral load, or proviral DNA frequency. Additional in vitro data suggest that the oxidant scavenger ascorbate also suppresses HIV replication in chronically and acutely infected T cells (129). Esta interaction appears to be synergistic when cells are exposed to ascorbate and N-acetylcysteine concurrently (128). In vivo studies with this combination have not been reported to date. Although oxidants may play a role in the pathogenesis of HIV infection, applying these findings for the development of potential therapeutic strategies in HIV-infected patients has been of limited benefit thus far. Oxidants may also be involved in the pathogenesis of other viral infections. As in HIV-infected cells, H2O2 effectively induces synthesis of viral antigens in several lymphoid cell lines that harbor the Epstein-Barr virus genome (234). Por el contrario, H2O2 markedly decreases the release of progeny hepatitis B virus (HBV) particles in cultured hepatoma cells without causing any significant difference in the overall pattern of host protein synthesis (333). These findings may be important in the pathophysiology of chronic HBV infection. In one circumstance, turning off viral gene expression may be a way for the host to eradicate HBV infection. However, this mechanism may allow the virus to evade complete destruction by shutting off viral expression in infected hepatocytes adjacent to an area of active inflammation. This would allow a few cells to escape antigen-specific killing and resume viral replication once the inflammation subsides. Levels of vitamin E in plasma are notably low in patients with chronic liver disease (306). Es widely established that vitamin E is an important cell membrane antioxidant which acts as a free radical scavenger. Uno might speculate that its deficiency in this setting may further perpetuate tissue damage caused by oxidant release from injured hepatocytes in patients with chronic viral hepatitis and ultimately with cirrhosis. However, there have been no controlled clinical trials assessing the therapeutic role of vitamin E in these patients. Human papillomavirus infection has been linked to an increased risk of acquiring human cervical carcinoma, and a recent study by Fernandez et al. suggests a potential oxidantdependent mechanism which could be involved (97). Ellos demonstrate that approximately 50% of healthy women possess polyamine oxidase and/or diamine oxidase in their cervical mucus. These enzymes were shown to act on spermine and spermidine (polyamines present in seminal fluid) to generate H2O2 and reactive aldehydes, which are likely to exert local mutagenic effects in vivo. These transformed cervical cells may exhibit prolonged survival in the presence of HPV infection through HPV suppression of apoptosis in the keratinocytes. Thus, the authors suggest that the effects of HPV infection of cervical cells may be synergistic with the effects of polyamine oxidation occurring in the cervical environment of sexually active women. The regulation of HPV replication may also be modified by oxidants, as the intracellular redox environment has been shown to affect the posttranslational DNA-binding activity of three E2 proteins (199). Virus-host cell interactions in relation to oxidant production also appear to be important in the pathogenesis of influenza A virus infection. Although neutrophils predominate in the early inflammatory response to influenza A virus (106), the ability of this virus to adversely affect neutrophil and monocyte function in infected patients is well established (133) and may contribute to secondary bacterial infections. The influenza A virus hemagglutinin molecule appears to be an important mediator in this process of abnormal leukocyte function (56, 131). Aunque exposure to the virus leads to neutrophil activation and generation of a respiratory burst, the neutrophil response is atypical with regard to calcium fluxes, phospholipase C activation, and release of H2O2 but not zO2 2 (130, 132). Daigneault et al. have further characterized this unique virus-phagocyte interplay, specifically through studies of the hemagglutininneutrophil receptor interaction (78). Clearly, further understanding of the role of oxidants in viral replication and virushost cell interactions for these and other viruses could potentially lead to new therapeutic interventions. UNTOWARD CONSEQUENCES OF OXIDANT PRODUCTION FOR THE HOST At sites of infection, host-derived oxidants not only place the offending organisms under oxidative stress but also cause stress to neighboring host tissues. As discussed above, these oxidants are derived primarily from phagocytes; however, they can be produced by other cell types inherently or via induction by redox-active agents. Tissue injury at sites of infection may be the result of the host inflammatory response to the pathogen rather than cytotoxic components of the microorganism. El role of oxidants in such processes will briefly be reviewed, given their intimate relationship with the pathophysiology of many infectious diseases. Readers are referred to the myriad of excellent recent reviews on oxidant-mediated tissue injury (76, 124-126, 209, 216, 309). Many aspects of acute and chronic inflammatory tissue injury appear to be mediated by oxidants released by neutrophils 10 MILLER AND BRITIGAN CLIN. MICROBIOL. REV. DOWNLOADED FROM cmr.ASM.ORG - July 20, 2010 and other phagocytes (216, 313). This process is enhanced by adherence of the phagocyte to the target cell surface (309). This adherence and subsequent movement of phagocytes from the blood to sites of inflammation require a complex signaling system involving a family of glycoproteins termed selectins. Selectins are synthesized by endothelial cells and stored in their secretory granules. When endothelial cells are activated by compounds such as thrombin or histamine (released in response to inflammation), the granules fuse with the outer membrane to expose the selectins on the cell surface. Phagocytes recognize these proteins, and this promotes their adherence to the endothelium and primes them for degranulation (190, 203). Following leukocyte activation, phagocyte-derived proteins termed integrins bind to their respective receptors on the endothelial cell. This interaction furt
er strengthens adhesion and directs the migration of the phagocyte beneath the endothelium. Thus, this process targets phagocytes to areas of inflammation, where, through the further recruitment of phagocytes, oxidant-mediated tissue injury may result. Phagocyte- derived H2O2 can also indirectly lead to inflammatory tissue injury by upregulating selectin expression on endothelial cells and promoting further neutrophil localization (238). Inflammatory tissue injury may also result via oxidant-induced cellular production of proinflammatory cytokines (72, 164, 206). Likewise, the production of these cytokines may potentiate further cellular oxidant release. Many of these interactions are mediated through the transcription-regulating factor, NF-kB (266). For example, in a rat model of neutrophilic alveolitis, endotoxin-induced NF-kB activation is thought to mediate the production of cytokine-induced neutrophil chemoattractant (analogous to human IL-8) by alveolar macrophages (25). This process is believed to be important for the recruitment of neutrophils and ultimately for the inflammatory tissue injury seen in this model. Oxidants can also activate NF-kB, promoting the production and release of cytokines such as IL-1 and TNF-a (8). Joint inflammation can also be induced by bacterial products, immune complexes, and crystals which recruit and activate phagocytes to form reactive oxygen species (125, 126) primarily. This process can result in tissue destruction via oxidant interactions with host proteoglycans, collagen, and elastin (124). Injury to pulmonary epithelial and pulmonary vascular endothelial cells can also occur as a consequence of microbial infection in the case of acute necrotizing pneumonia and chronic lung infection seen in cystic fibrosis patients (278, 309). A similar injury pattern can be observed with infection-related pulmonary complications such as acute respiratory distress syndrome and hyperoxic lung injury. The principal mechanism by which this lung injury occurs remains to be determined, but it appears to involve alterations in a number of parameters of epithelial and endothelial cell function inducible by phagocytederived zO2 2 and/or H2O2 (278). Iron-dependent formation of zOH appears to be involved in the ability of phagocytes to damage endothelial cells in vitro, with the endothelial cells serving as the source of catalytic iron (107, 175). Peroxynitrite also has been shown to inhibit pulmonary epithelial cell ion channels, suggesting that this species could contribute to diffusion barrier disruption under conditions in which both zO2 2 and NOz are present concurrently (14). MPO-derived oxidants released in response to a microbial stimulus may also contribute to inflammatory tissue injury directly via their toxic effects (148) and indirectly by their ability to inactivate serine protease inhibitors such as a1-antitrypsin (54, 83). These antiproteases play a critical role in limiting the activity of proteases such as human neutrophil elastase released at local sites of inflammation (202). Thus, protease inhibitor inactivation by MPO-derived oxidants may lead to emphysematous changes analogous to those seen in individuals congenitally deficient in a1-antitrypsin. Such processes have been hypothesized to contribute to lung injury associated with chronic bronchitis and other forms of chronic obstructive pulmonary disease (198, 233, 315). Others have suggested this process may also be involved in the lung disease observed in cystic fibrosis patients (214, 297). Data supporting a role for NOz and its derivatives in mediating inflammatory tissue injury in humans have been limited mainly to studies of autoimmune diseases (95, 216, 288). Evidencia supporting NOz production at sites of infection is lacking, however, as there are no definitive data demonstrating its formation by human phagocytes in vivo. In fact, recent literature suggests that NOz may also have antioxidant properties (163, 288, 319). However, bacterially derived lipopolysaccharide induces NOz production in endothelial cells. This process may contribute to the vasodilation and hypotension observed in septic shock (216). Like microorganisms, host cells have evolved a complex system to defend themselves against oxidant injury. As discussed above, eukaryotic cells synthesize CuZnSOD as a means of zO2 2 elimination. This enzyme is located in the cytosol and is usually constitutively expressed (12). Synthesis of a manganese- containing enzyme (MnSOD) can also be induced in the mitochondrial matrix under conditions of increased oxidative stress, specific cytokine stimulation, or heat shock (12). Desde the H2O2 formed by the dismutation of zO2 2 is also cytotoxic, eukaryotic cells have developed various mechanisms for its removal analogous to those found in prokaryotic microorganisms. This is accomplished by regulation of intracellular levels of catalase, the two GSH-dependent enzymes, GSH, and/or NADPH. Intra- and extracellular oxidant scavengers, such as ascorbic acid, vitamin E, b-carotene, and a-tocopherol, also probably play an important role in limiting cellular susceptibility to oxidant-mediated injury (313). Preventing the formation of zO2 2 and H2O2 is the primary mechanism by which cells can limit the formation of other potent oxidants such as zOH and the MPO-derived oxidants. Hydroxyl radical generation via the Haber-Weiss reaction can also be controlled by limiting the availability of redox-active iron catalysts through the formation of less active iron complexes such as extracellular lactoferrin and transferrin (7, 9, 42, 45, 320) and intracellular ferritin (10, 59). Heme oxygenase mRNA expression in mammalian cells is also known to be increased following cell exposure to oxidant stress. Although disputed by some investigators (231), the proposed mechanisms of protection afforded by heme oxygenase induction are twofold (6, 292). Heme oxygenase decreases the availability of intracellular iron capable of participating in the Haber-Weiss reaction by catalyzing the conversion of free heme to bile pigments. These bile pigments in turn exert antioxidant effects. Little is known about how host cells protect themselves from injury by NOz. It is likely that regulation of its production by the cell-specific NOz synthase will prove important. The extent of oxidant-mediated cytotoxicity observed at sites of inflammation is dependent on the balance between hostand microorganism-derived prooxidant and antioxidant forces. When this balance is swayed in favor of the prooxidants, not only microbial but also host cell cytotoxicity results, leading to clinical manifestations such as the sepsis syndrome, acute respiratory disease syndrome, lung destruction in diseases such as cystic fibrosis and a1-antitrypsin deficiency, and joint destruction in inflammatory arthritides. Further understanding of the mechanisms that regulate the prooxidant-antioxidant balance will probably have significant therapeutic implications in VOL. 10, 1997 ROLE OF OXIDANTS IN MICROBIAL PATHOPHYSIOLOGY 11 DOWNLOADED FROM cmr.ASM.ORG - July 20, 2010 the management of these and other diseases characterized by inflammatory tissue injury. CONCLUSIONES Defining the many roles of reactive oxygen species in hostmicrobial interactions has proved complex. Although these oxidants are consistent by-products of normal cellular metabolism, the concentration and potential biotoxicity can be markedly enhanced under conditions of exogenous oxidative stress, by exposure to pharmacologic agents, and, particularly, by phagocytes as a means of host defense against invading microorganisms. These oxidants can have beneficial and detrimental functions in both the host and the microorganism. Therefore, both have evolved complex adaptive mechanisms for protection against these compounds, including enzymatic and nonenzymatic oxidant-scavenging systems. These systems act as virulence factors for the microorganism which enable it to survive in a hostile environment. Despite the marked progress in this field recently, there are still many unanswered questions regarding the role of oxidants in microbial pathophysiology that will probably prove to be a promising research area in the futuro. ACKNOWLEDGMENTS This work was supported in part by awards from the VA Research Service and by NIH grants HL-44275, AI-28412, and AI-34954. Esta work was performed in part during the tenure of B. E. Britigan as an Established Investigator of the American Heart Association. R. A. Miller is supported through an NIH institutional training grant (AI- 07343). REFERENCIAS 1. Adams, L. B., J. B. Hibbs, Jr., R. R. Taintor, and J. L. Krahenbuhl. 1990. Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii. Role for synthesis of inorganic nitrogen oxides from L-arginine. J. Immunol. 144:2725-2729. 2. Albrich, J. M., J. H. Gilbaugh III, K. B. Callahan, and J. K. Hurst. 1986. Effects of the putative neutrophil-generated toxin, hypochlorous acid, on membrane permeability and transport systems of Escherichia coli. J. Clin. Invest. 78:177-184. 3. Almagor, M., I. Kahane, and S. Yatziv. 1984. Role of superoxide anion in host cell injury induced by Mycoplasma pneumoniae infection. 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