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By Human Neutrophils to Avoid Destruction Staphylococcus Aureus Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils This information is current as Jovanka M. Voyich, Kevin R. Braughton, Daniel E. of September 30, 2021. Sturdevant, Adeline R. Whitney, Battouli Saïd-Salim, Stephen F. Porcella, R. Daniel Long, David W. Dorward, Donald J. Gardner, Barry N. Kreiswirth, James M. Musser and Frank R. DeLeo J Immunol 2005; 175:3907-3919; ; Downloaded from doi: 10.4049/jimmunol.175.6.3907 http://www.jimmunol.org/content/175/6/3907 Supplementary http://www.jimmunol.org/content/suppl/2005/09/07/175.6.3907.DC1 http://www.jimmunol.org/ Material References This article cites 56 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/175/6/3907.full#ref-list-1 Why The JI? Submit online. by guest on September 30, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils1 Jovanka M. Voyich,* Kevin R. Braughton,* Daniel E. Sturdevant,* Adeline R. Whitney,* Battouli Saı¨d-Salim,§ Stephen F. Porcella,* R. Daniel Long,† David W. Dorward,‡ Donald J. Gardner,† Barry N. Kreiswirth,§ James M. Musser,¶ and Frank R. DeLeo2* Polymorphonuclear leukocytes (PMNs, or neutrophils) are critical for human innate immunity and kill most invading bacteria. However, pathogens such as Staphylococcus aureus avoid destruction by PMNs to survive, thereby causing human infections. The molecular mechanisms used by pathogens to circumvent killing by the immune system remain largely undefined. To that end, we studied S. aureus pathogenesis and bacteria-PMN interactions using strains originally isolated from individuals with community- acquired (CA) and hospital-acquired infections. Compared with strains from hospital infections (COL and MRSA252), strain MW2 and a methicillin-susceptible relative, MnCop, were significantly more virulent in a mouse model of S. aureus infection, and Downloaded from caused the greatest level of pathology in major vital organs. Although phagocytosis of each strain triggered production of reactive oxygen species and granule-phagosome fusion, those from CA infections were significantly more resistant to killing by human PMNs and caused greater host cell lysis. Microarray analysis of the strains during neutrophil phagocytosis identified genes comprising a global S. aureus response to human innate host defense. Genes involved in capsule synthesis, gene regulation, oxidative stress, and virulence, were up-regulated following ingestion of the pathogen. Notably, phagocytosis of strains from CA http://www.jimmunol.org/ infections induced changes in gene expression not observed in the other strains, including up-regulation of genes encoding viru- lence factors and hypothetical proteins. Our studies reveal a gene transcription program in a prominent human pathogen that likely contributes to evasion of innate host defense. The Journal of Immunology, 2005, 175: 3907–3919. olymorphonuclear leukocytes (PMNs,3 or neutrophils) are tizing pneumonia, endocarditis, and sepsis (1). Risk of S. aureus key effectors of the human innate immune system and pro- infection is increased during hospitalization, after surgery or dial- P vide critical defense against invading microorganisms. ysis, and in patients with indwelling percutaneous medical devices Phagocytosis of bacteria triggers production of reactive oxygen and/or catheters (1). Notably, there is an alarming increase in the species (ROS) and release of microbicidal granule components incidence of community-acquired (CA) S. aureus infections in by guest on September 30, 2021 into forming phagosomes. Although PMN-derived antimicrobial seemingly healthy individuals, a problem which underscores the products are highly effective at killing most bacteria, some patho- need to better understand virulence mechanisms (2). A number of gens evade destruction by neutrophils to survive and cause human factors are known to contribute to pathogenesis (1–6); however, a infections. An enhanced understanding of the molecular mecha- comprehensive analysis of changes in S. aureus gene expression nisms used by pathogens to circumvent the immune system is es- that occur during interaction with the human innate immune sys- sential for developing new treatments for infectious diseases. tem has not been performed. Furthermore, our understanding of Staphylococcus aureus is a leading cause of human infections the molecular mechanisms used by pathogens to avoid destruction worldwide. The pathogen causes a variety of diseases including by innate host defense is limited. To that end, we studied S. aureus impetigo, cellulitis, food poisoning, toxic shock syndrome, necro- pathogenesis and global gene expression during phagocytic inter- action with human PMNs. *Laboratory of Human Bacterial Pathogenesis, †Rocky Mountain Veterinary Branch, and ‡Rocky Mountain Microscopy Branch, Rocky Mountain Laboratories, National Materials and Methods Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Bacterial strains and culture MT 59840; §Public Health Research Institute Tuberculosis Center, International Cen- ¶ ter for Public Health, Newark, NJ 07103; and Center for Human Bacterial Patho- Staphylococcus aureus strains MW2 (5, 7), MnCop (8), LAC (9–12), genesis Research, Department of Pathology, Baylor University College of Medicine, MRSA252 (13), and COL (14) were selected carefully for this study based Houston, TX 70030 on several criteria, including the type of human disease caused by each Received for publication February 25, 2005. Accepted for publication July 5, 2005. (e.g., MW2 as the prototype for CA methicillin-resistant S. aureus The costs of publication of this article were defrayed in part by the payment of page (MRSA), and MRSA252 for its high prevalence in hospitals in the United charges. This article must therefore be hereby marked advertisement in accordance States and United Kingdom), available genome sequence information with 18 U.S.C. Section 1734 solely to indicate this fact. (MW2, MRSA252, and COL), historical significance (COL), available ge- 1 This work was supported in part by the Intramural Research Program of the National notype information (e.g., MLST, spa type, agr type, and SCCmec type), Institutes of Health, National Institute of Allergy and Infectious Diseases. and methicillin susceptibility (MnCop). Specifically, MW2 (pulsed-field type USA400) was originally isolated from a healthy 16-mo-old girl in 2 Address correspondence and reprint requests to Dr. Frank R. DeLeo, Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of North Dakota who died of septicemia and septic arthritis in 1998 (7, 15). Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840. LAC (pulsed-field type USA300) has caused recent S. aureus outbreaks in E-mail address: [email protected] athletes, many of which are skin infections (10, 11, 16). Recent reports also describe USA300 as the causative agent of staphylococcal necrotizing fas- 3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; ROS, reac- tive oxygen species; CA, community acquired; HA, hospital acquired; MRSA, me- ciitis (9) and severe community-onset pneumonia in healthy adults (12). thicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; DPBS, Dul- MnCop was originally isolated from an 8-mo-old infant who had S. aureus becco’s PBS; LDH, lactate dehydrogenase; TEM, transmission electron microscopy; toxic shock syndrome (8). Thus, each of the CA strains used in this study, GAS group A Streptococcus. i.e., MW2, MnCop, and LAC, has been linked to infections in individuals Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 3908 S. aureus EVADE KILLING BY HUMAN NEUTROPHILS with no known risk factors. In contrast, MRSA252 (pulsed-field type iden- bacteria:PMN. Plates were then centrifuged at 380 ϫ g for 8 min at 4°C to tical to EMRSA16), is the second leading cause of healthcare-associated S. synchronize phagocytosis (18). Samples were incubated at 37°C in a CO2 aureus infections in the United States (16). Descriptions of each strain are incubator for the indicated times (t ϭ 0 was processed immediately after summarized in Table I. centrifugation), medium was removed from the wells by aspiration, and S. aureus were grown in tryptic soy broth (BD Biosciences) containing cells were fixed on ice for 30 min with 4% paraformaldehyde. Fixative was 0.5% glucose. Bacterial cultures were inoculated from overnight cultures removed by aspiration, and uningested bacteria were counterstained with with a dilution of 1/200 for strains MW2, MnCop, and LAC, or 1/100
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