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1.  Seasonal H3N2 influenza A virus fails to enhance Staphylococcus aureus co-infection in a non-human primate respiratory tract infection model 
Virulence  2013;4(8):707-715.
Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.
PMCID: PMC3925702  PMID: 24104465
Staphylococcus aureus; influenza a virus; coinfection; USA300; MRSA; pneumonia
2.  Systems Biology and Innate Immunity 
Journal of innate immunity  2013;5(2):97-99.
PMCID: PMC3717263  PMID: 23428597
3.  Staphylococcus aureus Leukotoxin GH Promotes Inflammation 
The Journal of Infectious Diseases  2012;206(8):1185-1193.
Background. Staphylococcus aureus produces numerous molecules that facilitate survival in the host. We recently identified a novel S. aureus leukotoxin (leukotoxin GH [LukGH]) using proteomics, but its role in virulence remains unclear. Here we investigated the role of LukGH in vivo.
Methods. We tested cytotoxicity of LukGH toward polymorphonuclear leukocytes (PMNs) from mice, rabbits, monkeys, and humans. LukGH was administered to mice, rabbits, and a cynomolgus monkey by subcutaneous or intradermal injection to assess cytotoxicity or host response in vivo. The effects of LukGH in vivo were compared with those of Panton-Valentine leukocidin (PVL), a well-characterized S. aureus leukotoxin. The contribution of LukGH to S. aureus infection was tested using mouse and rabbit infection models.
Results. Susceptibility of PMNs to LukGH was similar between humans and cynomolgus monkeys, and was greater than that of rabbits, which in turn was greater than that of mice. LukGH or PVL caused skin inflammation in rabbits and a monkey, but deletion of neither lukGH nor lukGH and lukS/F-PV reduced severity of USA300 infections in rabbits or mice. Rather, some disease parameters (eg, rabbit abscess size) were increased following infection with a lukGH and lukS/F-PV deletion strain.
Conclusions. Our findings indicate that S. aureus leukotoxins enhance the host inflammatory response and influence the outcome of infection.
PMCID: PMC3448972  PMID: 22872735
4.  Staphylococcus aureus Protein A Promotes Immune Suppression 
mBio  2013;4(5):e00764-13.
Staphylococcus aureus is a prominent cause of human infections worldwide and is notorious for its ability to acquire resistance to antibiotics. Methicillin-resistant S. aureus (MRSA), in particular, is endemic in hospitals and is the most frequent cause of community-associated bacterial infections in the United States. Inasmuch as treatment options for severe MRSA infections are limited, there is need for a vaccine that protects against such infections. However, recent efforts to generate a staphylococcal vaccine have met with little success in human clinical trials. These failures are somewhat puzzling, since the vaccine antigens tested promote opsonophagocytosis in vitro and confer protection in animal infection models. One possibility is that the pathogen inhibits (and/or fails to elicit) the development of protective immunity in humans. Indeed, S. aureus produces numerous molecules that can potentially promote immune evasion, including protein A (SpA), an immunoglobulin (Ig)-binding protein present on the bacterial surface and freely secreted into the extracellular environment. SpA binds the Fc region of antibody and the Fab regions of the B-cell receptor, processes that are known to block opsonophagocytosis and cause B-cell death in vitro. In a recent study, Falugi et al. [F. Falugi, H. K. Kim, D. M. Missiakas, and O. Schneewind, mBio 4(5):e00575-13, 2013] showed that vaccination with spa mutant S. aureus strains lacking antibody Fc- and/or Fab-binding capacity protects against subsequent challenge with the USA300 epidemic strain. The findings provide strong support for the idea that SpA promotes S. aureus immune evasion in vivo and form the foundation for a new approach in our efforts to develop a vaccine that prevents severe S. aureus infections.
PMCID: PMC3791897  PMID: 24085782
5.  Community-associated methicillin-resistant Staphylococcus aureus 
Lancet  2010;375(9725):1557-1568.
Methicillin-resistant Staphylococcus aureus (MRSA) is endemic in hospitals worldwide and a significant cause of morbidity and mortality. Healthcare-associated MRSA infections occur in individuals with predisposing risk factors for disease, such as surgery or presence of an indwelling medical device. By contrast, community-associated MRSA (CA-MRSA) infections often occur in otherwise healthy individuals who lack such risk factors. In addition, CA-MRSA infections are epidemic in some countries. These observations suggest that CA-MRSA strains are more virulent and transmissible than traditional hospital-associated MRSA strains. Relatively limited treatment options for CA-MRSA infections compound the problem of enhanced virulence and transmission. Although progress has been made toward understanding emergence of CA-MRSA, virulence, and treatment of infections, our knowledge in these areas remains incomplete. Here were review the most current knowledge in these areas and provide perspective on future outlook for prophylaxis and/or new therapies for CA-MRSA infections.
PMCID: PMC3511788  PMID: 20206987
6.  Staphylococcus aureus survival in human blood 
Virulence  2011;2(6):567-569.
Methicillin-resistant Staphylococcus aureus (MRSA) is abundant in hospitals and in the United States is a leading cause of mortality due to infectious agents. Community-associated MRSA (CA-MRSA) strains such as USA300, which typically cause disease outside of healthcare settings, are also prevalent in the United States. Although most CA-MRSA infections affect skin and soft tissue, the pathogen can enter the bloodstream and ultimately cause severe disease. In a recent paper, we used USA300-specific microarrays to generate a comprehensive view of the molecules that facilitate S. aureus immune evasion and survival in human blood. Notably, genes encoding proteins involved in iron-uptake and utilization and gamma-hemolysin (hlgABC) are highly upregulated by USA300 during culture in human blood. Here we discuss the potential implication of these findings and the possible role of gammahemolysin in the success of S. aureus as a human pathogen.
PMCID: PMC3260549  PMID: 21971187
Staphylococcus aureus; MRSA; blood transcriptome; gammahemolysin; leukotoxin; neutrophil
7.  Comparative Analysis of USA300 Virulence Determinants in a Rabbit Model of Skin and Soft Tissue Infection 
The Journal of Infectious Diseases  2011;204(6):937-941.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are frequently associated with strains harboring genes encoding Panton-Valentine leukocidin (PVL). The role of PVL in the success of the epidemic CA-MRSA strain USA300 remains unknown. Here we developed a skin and soft tissue infection model in rabbits to test the hypothesis that PVL contributes to USA300 pathogenesis and compare it with well-established virulence determinants: alpha-hemolysin (Hla), phenol-soluble modulin-alpha peptides (PSMα), and accessory gene regulator (Agr). The data indicate that Hla, PSMα, and Agr contribute to the pathogenesis of USA300 skin infections in rabbits, whereas a role for PVL could not be detected.
PMCID: PMC3156927  PMID: 21849291
8.  Genomic Analysis of the Emergence of Vancomycin-Resistant Staphylococcus aureus 
mBio  2012;3(4):e00170-12.
Staphylococcus aureus is a human commensal bacterium and a prominent cause of infections globally. The high incidence of S. aureus infections is compounded by the ability of the microbe to readily acquire resistance to antibiotics. In the United States, methicillin-resistant S. aureus (MRSA) is a leading cause of morbidity and mortality by a single infectious agent. Therapeutic options for severe MRSA infections are limited to a few antibiotics to which the organism is typically susceptible, including vancomycin. Acquisition of high-level vancomycin resistance by MRSA is a major concern, but to date, there have been only 12 vancomycin-resistant S. aureus (VRSA) isolates reported in the United States and all belong to a phylogenetic lineage known as clonal complex 5. To gain enhanced understanding of the genetic characteristics conducive to the acquisition of vancomycin resistance by S. aureus, V. N. Kos et al. performed whole-genome sequencing of all 12 VRSA isolates and compared the DNA sequences to the genomes of other S. aureus strains. The findings provide new information about the evolutionary history of VRSA and identify genetic features that may bear on the relationship between S. aureus clonal complex 5 strains and the acquisition of vancomycin resistance genes from enterococci.
PMCID: PMC3388890  PMID: 22736541
9.  Multiplex PCR for Identification of Two Capsular Types in Epidemic KPC-Producing Klebsiella pneumoniae Sequence Type 258 Strains 
We developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258] cps-1 and cps-2) in epidemic Klebsiella pneumoniae ST258 strains. The assay performed with excellent sensitivity (100%) and specificity (100%) for identifying cps types in 60 ST258 K. pneumoniae sequenced isolates. The screening of 419 ST258 clonal isolates revealed a significant association between cps type and K. pneumoniae carbapenemase (KPC) variant: cps-1 is largely associated with KPC-2, while cps-2 is primarily associated with KPC-3.
PMCID: PMC4068549  PMID: 24733470
10.  Evolution of community- and healthcare-associated methicillin-resistant Staphylococcus aureus☆ 
Staphylococcus aureus is a prominent cause of human infections globally. The high prevalence of infections is compounded by antibiotic resistance—a significant problem for treatment. Methicillin-resistant S. aureus (MRSA) is endemic in hospitals and healthcare facilities worldwide, and is an increasingly common cause of community-associated bacterial infections in industrialized countries. Although much focus is placed on the role of S. aureus as a human pathogen, it is in fact a human commensal organism that has had a relatively long coexistence with the human host. Many S. aureus infections can be explained by host susceptibility or other predisposing risk factors. On the other hand, the emergence/re-emergence of successful S. aureus clones (referred to as epidemic waves) suggests a rapid bacterial adaption and evolution, which includes the emergence of antibiotic resistance and increased virulence and/or transmissibility. It is within this context that we review our understanding of selected S. aureus epidemic waves, and highlight the use of genome sequencing as a means to better understand the evolution of each lineage.
PMCID: PMC3884050  PMID: 23648426
Staphylococcus aureus; MRSA; Epidemic; Genome sequencing; Antimicrobial resistance
11.  Staphylococcus aureus LukGH promotes formation of neutrophil extracellular traps 
Staphylococcus aureus secretes numerous virulence factors that facilitate evasion of the host immune system. Among these molecules are pore-forming cytolytic toxins, including Panton-Valentine leukocidin (PVL), leukotoxins GH (LukGH; also known as LukAB) and DE (LukDE), and gamma-hemolysin (HlgABC). PVL and LukGH have potent cytolytic activity in vitro, and both toxins are proinflammatory in vivo. Although progress has been made towards elucidating the role of these toxins in S. aureus virulence, our understanding of the mechanisms that underly the proinflammatory capacity of these toxins, and the associated host response towards them, is incomplete. To address this deficiency in knowledge, we assessed the ability of LukGH to prime human PMNs for enhanced bactericidal activity and further investigated the impact of the toxin on neutrophil function. We found that unlike PVL, LukGH did not prime human neutrophils for increased production of reactive oxygen species nor did it enhance binding and/or uptake of S. aureus. Unexpectedly, LukGH promoted release of neutrophil extracellular traps (NETs), which in turn, ensnared but did not kill S. aureus. Furthermore, we found that electropermeabilization of human neutrophils—used as a separate means to create pores in the neutrophil plasma membrane—similarly induced formation of NETs, a finding consistent with the notion that NETs can form during non-specific cytolysis. We propose that the ability of LukGH to promote formation of NETs contributes to the inflammatory response and host defense against S. aureus infection.
PMCID: PMC3903389  PMID: 24190656
Staphylococcus aureus; neutrophil; leukotoxin; priming; extracellular trap
12.  A NET Outcome 
Neutrophils constitute a critical part of innate immunity and are well known for their ability to phagocytose and kill invading microorganisms. The microbicidal processes employed by neutrophils are highly effective at killing most ingested bacteria and fungi. However, an alternative non-phagocytic antimicrobial mechanism of neutrophils has been proposed whereby microorganisms are eliminated by neutrophil extracellular traps (NETs). NETs are comprised of DNA, histones, and antimicrobial proteins extruded by neutrophils during NETosis, a cell death pathway reported to be distinct from apoptosis, phagocytosis-induced cell death, and necrosis. Although multiple laboratories have reported NETs using various stimuli in vitro, the molecular mechanisms involved in this process have yet to be definitively elucidated, and many questions regarding the formation and putative role or function of NETs in innate host defense remain unanswered. It is with these questions in mind that we provide some reflection and perspective on NETs and NETosis.
PMCID: PMC3514450  PMID: 23227026
neutrophil; apoptosis; necrosis; phagocytosis; inflammation
13.  Targeting of alpha-hemolysin by active or passive immunization decreases severity of USA300 skin infections in a mouse model 
The Journal of infectious diseases  2010;202(7):1050-1058.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are predominantly those affecting skin and soft tissues. Although progress has been made, our knowledge of the molecules that contribute to the pathogenesis of CA-MRSA skin infections is incomplete. Here we tested the hypothesis that alpha-hemolysin (Hla) contributes to severity of USA300 skin infections in mice and determined whether vaccination against Hla reduces disease severity. Compared with wild-type USA300 and Newman strains, isogenic hla-negative (Δhla) strains caused significantly smaller skin lesions in a mouse infection model. Moreover, infection with wild-type strains produced dermonecrotic skin lesions, whereas there was little or no dermonecrosis in mice infected with Δhla strains. Passive immunization with Hla-specific antisera or active immunization with a non-toxigenic form of Hla significantly reduced the size of skin lesions caused by USA300 and prevented dermonecrosis. We conclude Hla is a potential target for therapeutics or vaccines designed to moderate severe S. aureus skin infections.
PMCID: PMC2945289  PMID: 20726702
alpha-hemolysin; MRSA; skin infection; Staphylococcus aureus; vaccine
14.  Rapid Neutrophil Destruction following Phagocytosis of Staphylococcus aureus 
Journal of Innate Immunity  2010;2(6):560-575.
Mechanisms underlying the enhanced virulence phenotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are incompletely defined, but presumably include evasion of killing by human polymorphonuclear leukocytes (PMNs or neutrophils). To better understand this phenomenon, we investigated the basis of rapid PMN lysis after phagocytosis of USA300, a prominent CA-MRSA strain. Survival of USA300 clinical isolates after phagocytosis ultimately resulted in neutrophil lysis. PMNs containing ingested USA300 underwent morphological changes consistent with apoptosis, but lysed rapidly thereafter (within 6 h), whereas cells undergoing FAS-mediated apoptosis or phagocytosis-induced cell death remained intact. Phagosome membranes remained intact until the point of PMN destruction, suggesting lysis was not caused by escape of S. aureus from phagosomes or the cytolytic action of pore-forming toxins. Microarray analysis of the PMN transcriptome after phagocytosis of representative community-associated S. aureus and healthcare-associated MRSA strains revealed changes unique to community-associated S. aureus strains, such as upregulation of transcripts involved in regulation of calcium homeostasis. Collectively, the data suggest that neutrophil destruction after phagocytosis of USA300 is in part a form of programmed necrosis rather than direct lysis by S. aureus pore-forming toxins. We propose that the ability of CA-MRSA strains to induce programmed necrosis of neutrophils is a component of enhanced virulence.
PMCID: PMC3219502  PMID: 20587998
Neutrophils; Bacterial infections; Host defense; Staphylococcus aureus
15.  Virulence of Endemic Nonpigmented Northern Australian Staphylococcus aureus Clone (Clonal Complex 75, S. argenteus) Is Not Augmented by Staphyloxanthin 
The Journal of Infectious Diseases  2013;208(3):520-527.
Staphylococcus aureus clonal complex 75 (herein referred to as S. argenteus) lacks the carotenoid pigment operon, crtOPQMN, responsible for production of the putative virulence factor, staphyloxanthin. Although a common cause of community-onset skin infections among Indigenous populations in northern Australia, this clone is infrequently isolated from hospital-based patients with either bacteremic or nonbacteremic infections. We hypothesized that S. argenteus would have attenuated virulence compared to other S. aureus strains due to its staphyloxanthin “deficiency.” Compared to prototypical S. aureus strains, S. argenteus was more susceptible to oxidative stress and neutrophil killing in vitro and had reduced virulence in murine sepsis and skin infection models. Transformation with pTX-crtOPQMN resulted in staphyloxanthin expression and increased resistance to oxidative stress in vitro. However, neither resistance to neutrophil killing nor in vivo virulence was increased. Thus, reduced virulence of S. argenteus in these models is due to mechanisms unrelated to lack of staphyloxanthin production.
PMCID: PMC3699000  PMID: 23599317
Staphylococcus aureus; staphyloxanthin; virulence; Australia; carotenoid pigment
16.  Host Defense and Pathogenesis in Staphylococcus aureus Infections 
Staphylococcus aureus is the most abundant cause of bacterial infections in the United States. As such, the pathogen has devised means to circumvent destruction by the innate immune system. Neutrophils are a critical component of innate immunity and the primary cellular defense against S. aureus infections. Herein we review human neutrophil function in the context of S. aureus virulence mechanisms, and provide an overview of community-associated methicillin resistant S. aureus (CA-MRSA) pathogenicity.
PMCID: PMC2748223  PMID: 19135914
staphylococcus; neutrophil; innate immunity; virulence
17.  Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era  
The Journal of Clinical Investigation  2009;119(9):2464-2474.
Staphylococcus aureus is the leading cause of bacterial infections in developed countries and produces a wide spectrum of diseases, ranging from minor skin infections to fatal necrotizing pneumonia. Although S. aureus infections were historically treatable with common antibiotics, emergence of drug-resistant organisms is now a major concern. Methicillin-resistant S. aureus (MRSA) was endemic in hospitals by the late 1960s, but it appeared rapidly and unexpectedly in communities in the 1990s and is now prevalent worldwide. This Review focuses on progress made toward understanding the success of community-associated MRSA as a human pathogen, with an emphasis on genome-wide approaches and virulence determinants.
PMCID: PMC2735934  PMID: 19729844
18.  Epidemic Klebsiella pneumoniae ST258 Is a Hybrid Strain 
mBio  2014;5(3):e01355-14.
Carbapenem-resistant Enterobacteriaceae (CRE), especially Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, pose an urgent threat in health facilities in the United States and worldwide. K. pneumoniae isolates classified as sequence type 258 (ST258) by multilocus sequence typing are largely responsible for the global spread of KPC. A recent comparative genome study revealed that ST258 K. pneumoniae strains are two distinct genetic clades; however, the molecular origin of ST258 largely remains unknown, and our understanding of the evolution of the two genetic clades is incomplete. Here we compared the genetic structures and single-nucleotide polymorphism (SNP) distributions in the core genomes of strains from two ST258 clades and other STs (ST11, ST442, and ST42). We identified an ~1.1-Mbp region on ST258 genomes that is homogeneous to that of ST442, while the rest of the ST258 genome resembles that of ST11. Our results suggest ST258 is a hybrid clone—80% of the genome originated from ST11-like strains and 20% from ST442-like strains. Meanwhile, we sequenced an ST42 strain that carries the same K-antigen-encoding capsule polysaccharide biosynthesis gene (cps) region as ST258 clade I strains. Comparison of the cps-harboring regions between the ST42 and ST258 strains (clades I and II) suggests the ST258 clade I strains evolved from a clade II strain as a result of cps region replacement. Our findings unravel the molecular evolution history of ST258 strains, an important first step toward the development of diagnostic, therapeutic, and vaccine strategies to combat infections caused by multidrug-resistant K. pneumoniae.
Recombination events and replacement of chromosomal regions have been documented in various bacteria, and these events have given rise to successful pathogenic clones. Here we used comparative genomic analyses to discover that the ST258 K. pneumoniae genome is a hybrid—80% of the chromosome is homologous to ST11 strains, while the remaining 20% is homologous to that of ST442. Meanwhile, a recent study indicated that ST258 strains can be segregated into two ST258 clades, with distinct capsule polysaccharide gene (cps) regions. Our analysis suggests ST258 clade I strains evolved from clade II through homologous recombination of cps region. Horizontal transfer of the cps region appears to be a key element driving the molecular diversification in K. pneumoniae strains. These findings not only extend our understanding of the molecular evolution of ST258 but are an important step toward the development of effective control and treatment strategies for multidrug-resistant K. pneumoniae.
PMCID: PMC4073492  PMID: 24961694
19.  Panton-Valentine leukocidin is not a virulence determinant in murine models of community-associated methicillin-resistant Staphylococcus aureus disease 
The Journal of infectious diseases  2008;198(8):1166-1170.
Increases in the incidence and severity of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have spawned efforts to define unique virulence properties among prevalent strains. Panton-Valentine leukocidin (PVL), a pore-forming cytotoxin, has garnered attention due to its epidemiologic association with CA-MRSA. Using the clinical isolate LAC, representative of the epidemic USA300 strain, and its isogenic PVL-negative strain in murine models of staphylococcal skin infection and pneumonia, we have extended recent studies by assessing the contribution of PVL in the BALB/c genetic background. The data herein support the observation that PVL does not contribute to the pathogenesis of staphylococcal infection of mice.
PMCID: PMC2574921  PMID: 18729780
Community-associated methicillin resistant Staphylococcus aureus (CA-MRSA); Panton-Valentine leukocidin (PVL); USA300; skin infection; pneumonia; animal models
20.  Selective Chemical Inhibition of agr Quorum Sensing in Staphylococcus aureus Promotes Host Defense with Minimal Impact on Resistance 
PLoS Pathogens  2014;10(6):e1004174.
Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.
Author Summary
New approaches are needed to lessen the burden of antibiotic resistant bacterial infections. One strategy is to develop therapies that target virulence which rely on host defense elements to clear the bacteria rather than direct antimicrobial killing. Quorum sensing is a bacterial signaling mechanism that often regulates virulence in medically relevant bacterial pathogens. Therefore, drugs that inhibit quorum sensing can promote host defense by rendering the pathogenic bacteria avirulent and/or less fit for survival within the host. Our work addressed this strategy in the pathogen Staphylococcus aureus which is the major cause of acute bacterial skin and soft tissue infections. We conducted a high throughput screen to identify compounds that could inhibit signaling by the quorum sensing operon, agr. We found a compound that we termed savirin (S. aureus virulence inhibitor) that could inhibit signaling by this operon. The drug helped the innate immune system in animals to clear bacteria that express this operon without affecting clearance of bacteria that do not have this operon. We addressed the mechanism of action of this compound and whether resistance or tolerance to this compound would likely develop. Our data indicate for the first time that host defense against S. aureus skin infections can be enhanced by chemical inhibition of agr-mediated quorum sensing.
PMCID: PMC4055767  PMID: 24945495
21.  Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis 
Journal of innate immunity  2012;5(2):124-136.
We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24 h time period relative to the uninfected controls. Of ~47,000 genes analyzed, 3,435 were significantly up- or down-regulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes associated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2, and CASP8) and genes that act via the NF B pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2, and RELA, IL1B, CAST, CDK2, GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that X-linked inhibitor of apoptosis (XIAP) protein remained abundant in PMNs over 48 h of LVS infection, whereas BAX mRNA and protein were progressively down-regulated. These data strongly suggest that antiapoptotic and pro-survival mechanisms collaborate to sustain the viability of F. tularensis infected neutrophils.
PMCID: PMC3703778  PMID: 22986450
neutrophil; polymorphonuclear leukocyte; apoptosis; gene expression; microarray; tularemia; XIAP; BAX
22.  Towards a comprehensive understanding of the role of neutrophils in innate immunity: a systems biology-level approach 
The innate immune system is the first line of host defense against invading microorganisms. Polymorphonuclear leukocytes (PMNs or neutrophils) are the most abundant leukocyte in humans and essential to the innate immune response against invading pathogens. Compared to the acquired immune response, which requires time to develop and is dependent on previous interaction with specific microbes, the ability of neutrophils to kill microorganisms is immediate, non-specific, and not dependent on previous exposure to microorganisms. Historically, studies of PMN-pathogen interaction focused on the events leading to killing of microorganisms, such as recruitment/chemotaxis, transmigration, phagocytosis, and activation, whereas post-phagocytosis sequelae were infrequently considered. In addition, it was widely accepted that human neutrophils possessed limited capacity for new gene transcription and thus, relatively little biosynthetic capacity. This notion has changed dramatically within the past decade. Further, there is now more effort directed to understand the events occurring in PMNs after killing of microbes. Herein we review the systems biology-level approaches that have been used to gain an enhanced view of the role of neutrophils during host-pathogen interaction. We anticipate that these and future systems-level studies will ultimately will provide information critical to our understanding, treatment, and control of diseases caused by pathogenic microorganisms.
PMCID: PMC3501127  PMID: 20836000
Neutrophil; phagocytosis; microarray; inflammation; apoptosis
23.  Toward an Understanding of the Evolution of Staphylococcus aureus Strain USA300 during Colonization in Community Households 
Genome Biology and Evolution  2012;4(12):1275-1285.
Staphylococcus aureus is a frequent cause of serious infections and also a human commensal. The emergence of community-associated methicillin-resistant S. aureus led to a dramatic increase in skin and soft tissue infections worldwide. This epidemic has been driven by a limited number of clones, such as USA300 in the United States. To better understand the extent of USA300 evolution and diversification within communities, we performed comparative whole-genome sequencing of three clinical and five colonizing USA300 isolates collected longitudinally from three unrelated households over a 15-month period. Phylogenetic analysis that incorporated additional geographically diverse USA300 isolates indicated that all but one likely arose from a common recent ancestor. Although limited genetic adaptation occurred over the study period, the greatest genetic heterogeneity occurred between isolates from different households and within one heavily colonized household. This diversity allowed for a more accurate tracking of interpersonal USA300 transmission. Sequencing of persisting USA300 isolates revealed mutations in genes involved in major aspects of S. aureus function: adhesion, cell wall biosynthesis, virulence, and carbohydrate metabolism. Genetic variations also included accumulation of multiple polymorphisms within select genes of two multigene operons, suggestive of small genome rearrangements rather than de novo single point mutations. Such rearrangements have been underappreciated in S. aureus and may represent novel means of strain variation. Subtle genetic changes may contribute to USA300 fitness and persistence. Elucidation of small genome rearrangements reveals a potentially new and intriguing mechanism of directed S. aureus genome diversification in environmental niches and during pathogen–host interactions.
PMCID: PMC3542572  PMID: 23104992
evolution; genome rearrangement; repeat deletions
24.  Innate Immunity against Granulibacter bethesdensis, an Emerging Gram-Negative Bacterial Pathogen 
Infection and Immunity  2012;80(3):975-981.
Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H2O2 but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O2-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.
PMCID: PMC3294668  PMID: 22184421
25.  Presence of Genes Encoding Panton-Valentine Leukocidin Is Not the Primary Determinant of Outcome in Patients with Hospital-Acquired Pneumonia Due to Staphylococcus aureus 
Journal of Clinical Microbiology  2012;50(3):848-856.
The impact of Panton-Valentine leukocidin (PVL) on the outcome in Staphylococcus aureus pneumonia is controversial. We genotyped S. aureus isolates from patients with hospital-acquired pneumonia (HAP) enrolled in two registrational multinational clinical trials for the genetic elements carrying pvl and 30 other virulence genes. A total of 287 isolates (173 methicillin-resistant S. aureus [MRSA] and 114 methicillin-susceptible S. aureus [MSSA] isolates) from patients from 127 centers in 34 countries for whom clinical outcomes of cure or failure were available underwent genotyping. Of these, pvl was detected by PCR and its product confirmed in 23 isolates (8.0%) (MRSA, 18/173 isolates [10.4%]; MSSA, 5/114 isolates [4.4%]). The presence of pvl was not associated with a higher risk for clinical failure (4/23 [17.4%] versus 48/264 [18.2%]; P = 1.00) or mortality. These findings persisted after adjustment for multiple potential confounding variables. No significant associations between clinical outcome and (i) presence of any of the 30 other virulence genes tested, (ii) presence of specific bacterial clone, (iii) levels of alpha-hemolysin, or (iv) delta-hemolysin production were identified. This study suggests that neither pvl presence nor in vitro level of alpha-hemolysin production is the primary determinant of outcome among patients with HAP caused by S. aureus.
PMCID: PMC3295120  PMID: 22205797

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