Biomedical subjects

Kevin R Braughton

Publications and source records attributed to Kevin R Braughton.

10 recordsLinked to original sources

PubMed2006

Is Panton-Valentine leukocidin the major virulence determinant in community-associated methicillin-resistant Staphylococcus aureus disease?

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major problem in hospitals, and it is now spreading in the community. A single toxin, Panton-Valentine leukocidin (PVL), has been linked by epidemiological studies to community-associated MRSA (CA-MRSA) disease. However, the role that PVL plays in the pathogenesis of CA-MRSA has not been tested directly. To that end, we used mouse infection models to compare the virulence of PVL-positive with that of PVL-negative CA-MRSA representing the leading disease-causing strains. Unexpectedly, strains lacking PVL were as virulent in mouse sepsis and abscess models as those containing the leukotoxin. Isogenic PVL-negative (lukS/F-PV knockout) strains of USA300 and USA400 were as lethal as wild-type strains in a sepsis model, and they caused comparable skin disease. Moreover, lysis of human neutrophils and pathogen survival after phagocytosis were similar between wild-type and mutant strains. Although the toxin may be a highly linked epidemiological marker for CA-MRSA strains, we conclude that PVL is not the major virulence determinant of CA-MRSA.

Abscess↗

PubMed2005

Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils.

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 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 infections induced changes in gene expression not observed in the other strains, including up-regulation of genes encoding virulence 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.

Gene Expression Profiling↗

PubMed2004

Engagement of the pathogen survival response used by group A Streptococcus to avert destruction by innate host defense.

Neutrophils are a critical component of human innate host defense and efficiently kill the vast majority of invading microorganisms. However, bacterial pathogens such as group A Streptococcus (GAS) successfully avert destruction by neutrophils to cause human infections. Relatively little is known about how pathogens detect components of the innate immune system to respond and survive within the host. In this study, we show that inactivation of a two-component gene regulatory system designated Ihk-Irr significantly attenuates streptococcal virulence in mouse models of soft tissue infection and bacteremia. Microarray analysis of wild-type and irr-negative mutant (irr mutant) GAS strains revealed that Ihk-Irr influenced expression of 20% of all transcripts in the pathogen genome. Notably, at least 11 genes involved in cell wall synthesis, turnover, and/or modification were down-regulated in the irr mutant strain. Compared with the wild-type strain, significantly more of the irr mutant strain was killed by human neutrophil components that destroy bacteria by targeting the cell envelope (cell wall and/or membrane). Unexpectedly, expression of ihk and irr was dramatically increased in the wild-type strain exposed to these same neutrophil products under conditions that favored cell envelope damage. We report a GAS mechanism for detection of innate host defense that initiates the pathogen survival response, in which cell wall synthesis is critical. Importantly, our studies identify specific genes in the pathogen survival response as potential targets to control human infections.

Animals↗

PubMed2004

Gene expression profiling provides insight into the pathophysiology of chronic granulomatous disease.

Human polymorphonuclear leukocytes (PMNs or neutrophils) kill invading microorganisms with reactive oxygen species (ROS) and cytotoxic granule components. PMNs from individuals with X-linked chronic granulomatous disease (XCGD) do not produce ROS, thereby rendering these individuals more susceptible to infection. In addition, XCGD patients develop tissue granulomas that obstruct vital organs, the mechanism(s) for which are unknown. To gain insight into the molecular processes that contribute to the pathophysiology of XCGD, including formation of granulomas, we compared global gene expression in PMNs from XCGD patients and healthy control individuals. Genes encoding mediators of inflammation and host defense, including CD11c, CD14, CD54, FcgammaR1, FcalphaR, CD120b, TLR5, IL-4R, CCR1, p47(phox), p40(phox), IL-8, CXCL1, Nramp1, and calgranulins A and B, were up-regulated constitutively in unstimulated XCGD patient PMNs. By comparing transcript levels in normal and XCGD PMNs after phagocytosis, we discovered 206 genes whose expression changed in the presence and the absence of ROS, respectively. Notably, altered Bcl2-associated X protein synthesis accompanied defective neutrophil apoptosis in XCGD patients. We hypothesize that granuloma formation in XCGD patients reflects both increased proinflammatory activity and defective PMN apoptosis, and we conclude that ROS contribute directly or indirectly to the resolution of the inflammatory response by influencing PMN gene transcription.

Adolescent↗

PubMed2004

Polysaccharide intercellular adhesin (PIA) protects Staphylococcus epidermidis against major components of the human innate immune system.

The skin commensal and opportunistic pathogen Staphylococcus epidermidis is the leading cause of nosocomial and biofilm-associated infections. Little is known about the mechanisms by which S. epidermidis protects itself against the innate human immune system during colonization and infection. We used scanning electron microscopy to demonstrate that the exopolysaccharide intercellular adhesin (PIA) resides in fibrous strands on the bacterial cell surface, and that lack of PIA production results in complete loss of the extracellular matrix material that has been suggested to mediate immune evasion. Phagocytosis and killing by human polymorphonuclear leucocytes was significantly increased in a mutant strain lacking PIA production compared with the wild-type strain. The mutant strain was also significantly more susceptible to killing by major antibacterial peptides of human skin, cationic human beta-defensin 3 and LL-37, and anionic dermcidin. PIA represents the first defined factor of the staphylococcal biofilm matrix that protects against major components of human innate host defence.

Humans↗

PubMed2003

Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils.

Human polymorphonuclear leukocytes (PMNs or neutrophils) are essential to the innate immune response against bacterial pathogens. Recent evidence suggests that PMN apoptosis facilitates resolution of inflammation during bacterial infection. Although progress has been made toward understanding apoptosis in neutrophils, very little is known about transcriptional regulation of this process during bacterial infection. To gain insight into the molecular processes that facilitate resolution of infection, we measured global changes in PMN gene expression during phagocytosis of a diverse group of bacterial pathogens. Genes encoding key effectors of apoptosis were up-regulated, and receptors critical to innate immune function were down-regulated during apoptosis induced by phagocytosis of Burkholderia cepacia, Borrelia hermsii, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus pyogenes. Importantly, we identified genes that comprise a common apoptosis differentiation program in human PMNs after phagocytosis of pathogenic bacteria. Unexpectedly, phagocytosis of Str. pyogenes induced changes in neutrophil gene expression not observed with other pathogens tested, including down-regulation of 21 genes involved in responses to IFN. Compared with other bacteria, PMN apoptosis was significantly accelerated by Str. pyogenes and was followed by necrosis. Thus, we hypothesize that there are two fundamental outcomes for the interaction of bacterial pathogens with neutrophils: (i) phagocytosis of bacteria induces an apoptosis differentiation program in human PMNs that contributes to resolution of bacterial infection, or (ii) phagocytosis of microorganisms such as Str. pyogenes alters the apoptosis differentiation program in neutrophils, resulting in pathogen survival and disease.

Apoptosis↗

PubMed2003

Down-regulation of proinflammatory capacity during apoptosis in human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) are essential to innate immunity in humans and contribute significantly to inflammation. Although progress has been made, the molecular basis for termination of inflammation in humans is incompletely characterized. We used human oligonucleotide microarrays to identify genes encoding inflammatory mediators that were differentially regulated during the induction of apoptosis. One hundred thirty-three of 212 differentially expressed genes encoding proinflammatory factors, signal transduction mediators, adhesion molecules, and other proteins that facilitate the inflammatory response were down-regulated during the induction of apoptosis following PMN phagocytosis. Among these, 42 genes encoded proteins critical to the inflammatory response, including receptors for IL-8 beta, IL-10 alpha, IL-13 alpha 1, IL-15 alpha, IL-17, IL-18, C1q, low-density lipoprotein, IgG Fc (CD32), and formyl peptide, Toll-like receptor 6, platelet/endothelial cell adhesion molecule-1 (CD31), P-selectin (CD62), IL-1 alpha, IL-16, and granulocyte chemoattractant protein-2 were down-regulated. Many of these genes were similarly down-regulated during Fas-mediated or camptothecin-induced apoptosis. We used flow cytometry to confirm that IL-8R beta (CXCR2) and IL-1 alpha were significantly down-regulated during PMN apoptosis. We also discovered that 23 genes encoding phosphoinositide and calcium-mediated signal transduction components, which comprise complex pathways essential to the inflammatory response of host cells, were differentially regulated during PMN apoptosis. Importantly, our data demonstrate that PMNs down-regulate proinflammatory capacity at the level of gene expression during induction of apoptosis. These findings provide new insight into the molecular events that resolve inflammation following PMN activation in humans.

Apoptosis↗

PubMed2003

Genome-wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes.

Group A Streptococcus (GAS) evades polymorphonuclear leukocyte (PMN) phagocytosis and killing to cause human disease, including pharyngitis and necrotizing fasciitis (flesh-eating syndrome). We show that GAS genes differentially regulated during phagocytic interaction with human PMNs comprise a global pathogen-protective response to innate immunity. GAS prophage genes and genes involved in virulence, oxidative stress, cell wall biosynthesis, and gene regulation were up-regulated during PMN phagocytosis. Genes encoding novel secreted proteins were up-regulated, and the proteins were produced during human GAS infections. We discovered an essential role for the Ihk-Irr two-component regulatory system in evading PMN-mediated killing and promoting host-cell lysis, processes that would facilitate GAS pathogenesis. Importantly, the irr gene was highly expressed during human GAS pharyngitis. We conclude that a complex pathogen genetic program circumvents human innate immunity to promote disease. The gene regulatory program revealed by our studies identifies previously undescribed potential vaccine antigens and targets for therapeutic interventions designed to control GAS infections.

Gene Expression Regulation, Bacterial↗

PubMed2003

An apoptosis-differentiation program in human polymorphonuclear leukocytes facilitates resolution of inflammation.

Human polymorphonuclear leukocytes (PMNs) are an essential part of innate immunity and contribute significantly to inflammation. Although much is understood about the inflammatory response, the molecular basis for termination of inflammation in humans is largely undefined. We used human oligonucleotide microarrays to identify genes differentially regulated during the onset of apoptosis occurring after PMN phagocytosis. Genes encoding proteins that regulate cell metabolism and vesicle trafficking comprised 198 (98 genes induced, 100 genes repressed) of 867 differentially expressed genes. We discovered that complex cellular pathways involving glutathione and thioredoxin detoxification systems, heme catabolism, ubiquitin-proteasome degradation, purine nucleotide metabolism, and nuclear import were regulated at the level of gene expression during the initial stages of PMN apoptosis. Eleven genes encoding key regulators of glycolysis, the hexose monophosphate shunt, the glycerol-phosphate shuttle, and oxidative phosphorylation were induced. Increased levels of cellular reduced glutathione and gamma-glutamyltransferase and glycolytic activity confirmed that several of these metabolic pathways were up-regulated. In contrast, seven genes encoding critical enzymes involved in fatty acid beta-oxidation, which can generate toxic lipid peroxides, were down-regulated. Our results indicate that energy metabolism and oxidative stress-response pathways are gene-regulated during PMN apoptosis. We propose that changes in PMN gene expression leading to programmed cell death are part of an apoptosis-differentiation program, a final stage of transcriptionally regulated PMN maturation that is accelerated significantly by phagocytosis. These findings provide new insight into the molecular events that contribute to the resolution of inflammation in humans.

Apoptosis↗

PubMed2002

Opsonophagocytosis-inhibiting mac protein of group a streptococcus: identification and characteristics of two genetic complexes.

Recently, it was reported that a streptococcal Mac protein (designated Mac(5005)) made by serotype M1 group A Streptococcus (GAS) is a homologue of human CD11b that inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear leukocytes (PMNs) (B. Lei, F. R. DeLeo, N. P. Hoe, M. R. Graham, S. M. Mackie, R. L. Cole, M. Liu, H. R. Hill, D. E. Low, M. J. Federle, J. R. Scott, and J. M. Musser, Nat. Med. 7:1298-1305, 2001). To study mac variation and expression of the Mac protein, the gene in 67 GAS strains representing 36 distinct M protein serotypes was sequenced. Two distinct genetic complexes were identified, and they were designated complex I and complex II. Mac variants in each of the two complexes were closely related, but complex I and complex II variants differed on average at 50.66 +/- 5.8 amino acid residues, most of which were located in the middle one-third of the protein. Complex I Mac variants have greater homology with CD11b than complex II variants. GAS strains belonging to serotypes M1 and M3, the most abundant M protein serotypes responsible for human infections in many case series, have complex I Mac variants. The mac gene was cloned from representative strains assigned to complexes I and II, and the Mac proteins were purified to apparent homogeneity. Both Mac variants had immunoglobulin G (IgG)-endopeptidase activity. In contrast to Mac(5005) (complex I), Mac(8345) (complex II) underwent autooxidation of its cysteine residues, resulting in the loss of IgG-endopeptidase activity. A Mac(5005) Cys94Ala site-specific mutant protein was unable to cleave IgG but retained the ability to inhibit IgG-mediated phagocytosis by human PMNs. Thus, the IgG-endopeptidase activity was not essential for the key biological function of Mac(5005). Although Mac(5005) and Mac(8345) each have an Arg-Gly-Asp (RGD) motif, the proteins differed in their interactions with human integrins alpha(v)beta(3) and alpha(IIb)beta(3). Binding of Mac(5005) to integrins alpha(v)beta(3) and alpha(IIb)beta(3) was mediated primarily by the RGD motif in Mac(5005), whereas binding of Mac(8345) involved the RGD motif and a region in the middle one-third of the molecule whose sequence is different in Mac(8345) and Mac(5005). Taken together, the data add to the emerging theme in GAS pathogenesis that allelic variation in virulence genes contributes to fundamental differences in host-pathogen interactions among strains.

Amino Acid Sequence↗