Alpha-Toxin Induces Programmed Cell Death of Human T cells, B cells, and Monocytes during USA300 Infection

This investigation examines the influence of alpha-toxin (Hla) during USA300 infection of human leukocytes. Survival of an USA300 isogenic deletion mutant of hla (USA300Δhla) in human blood was comparable to the parental wild-type strain and polymorphonuclear leukocyte (PMN) plasma membrane permeability caused by USA300 did not require Hla. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) following infection by USA300, USA300Δhla, and USA300Δhla transformed with a plasmid over-expressing Hla (USA300Δhla Comp) demonstrated this toxin plays a significant role inducing plasma membrane permeability of CD14+, CD3+, and CD19+ PBMCs. Rapid plasma membrane permeability independent of Hla was observed for PMNs, CD14+ and CD19+ PBMCs following intoxication with USA300 supernatant while the majority of CD3+ PBMC plasma membrane permeability induced by USA300 required Hla. Addition of recombinant Hla to USA300Δhla supernatant rescued CD3+ and CD19+ PBMC plasma membrane permeability generated by USA300 supernatant. An observed delay in plasma membrane permeability caused by Hla in conjunction with Annexin V binding and ApoBrdU Tunel assays examining PBMCs intoxicated with recombinant Hla or infected with USA300, USA300Δhla, USA300Δhla Comp, and USA300ΔsaeR/S suggest Hla induces programmed cell death of monocytes, B cells, and T cells that results in plasma membrane permeability. Together these findings underscore the importance of Hla during S. aureus infection of human tissue and specifically demonstrate Hla activity during USA300 infection triggers programmed cell death of human monocytes, T cells and B cells that leads to plasma membrane permeability

Importance of the Global Regulators Agr and SaeRS in the Pathogenesis of CA-MRSA USA300 Infection

CA-MRSA infection, driven by the emergence of the USA300 genetic background, has become epidemic in the United States. USA300 isolates are hypervirulent, compared with other CA- and HA-MRSA strains, in experimental models of necrotizing pneumonia and skin infection. Interestingly, USA300 isolates also have increased expression of core genomic global regulatory and virulence factor genes, including agr and saeRS. To test the hypothesis that agr and saeRS promote the observed hypervirulent phenotype of USA300, isogenic deletion mutants of each were constructed in USA300. The effects of gene deletion on expression and protein abundance of selected downstream virulence genes were assessed by semiquantitative real-time reverse-transcriptase PCR (qRT-PCR) and western blot, respectively. The effects of gene deletion were also assessed in mouse models of necrotizing pneumonia and skin infection. Deletion of saeRS, and, to a lesser extent, agr, resulted in attenuated expression of the genes encoding α-hemolysin (hla) and the Panton-Valentine leukocidin (lukSF-PV). Despite the differences in hla transcription, the toxin was undetectable in culture supernatants of either of the deletion mutants. Deletion of agr, but not saeRS, markedly increased the expression of the gene encoding protein A (spa), which correlated with increased protein abundance. Each deletion mutant demonstrated significant attenuation of virulence, compared with wild-type USA300, in mouse models of necrotizing pneumonia and skin infection. We conclude that agr and saeRS each independently contribute to the remarkable virulence of USA300, likely by means of their effects on expression of secreted toxins

Characterization and Comparison of 2 Distinct Epidemic Community-Associated Methicillin-Resistant Staphylococcus aureus Clones of ST59 Lineage.

Sequence type (ST) 59 is an epidemic lineage of community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) isolates. Taiwanese CA-MRSA isolates belong to ST59 and can be grouped into 2 distinct clones, a virulent Taiwan clone and a commensal Asian-Pacific clone. The Taiwan clone carries the Panton-Valentine leukocidin (PVL) genes and the staphylococcal chromosomal cassette mec (SCCmec) VT, and is frequently isolated from patients with severe disease. The Asian-Pacific clone is PVL-negative, carries SCCmec IV, and a frequent colonizer of healthy children. Isolates of both clones were characterized by their ability to adhere to respiratory A549 cells, cytotoxicity to human neutrophils, and nasal colonization of a murine and murine sepsis models. Genome variation was determined by polymerase chain reaction of selected virulence factors and by multi-strain whole genome microarray. Additionally, the expression of selected factors was compared between the 2 clones. The Taiwan clone showed a much higher cytotoxicity to the human neutrophils and caused more severe septic infections with a high mortality rate in the murine model. The clones were indistinguishable in their adhesion to A549 cells and persistence of murine nasal colonization. The microarray data revealed that the Taiwan clone had lost the ø3-prophage that integrates into the β-hemolysin gene and includes staphylokinase- and enterotoxin P-encoding genes, but had retained the genes for human immune evasion, scn and chps. Production of the virulence factors did not differ significantly in the 2 clonal groups, although more α-toxin was expressed in Taiwan clone isolates from pneumonia patients. In conclusion, the Taiwan CA-MRSA clone was distinguished by enhanced virulence in both humans and an animal infection model. The evolutionary acquisition of PVL, the higher expression of α-toxin, and possibly the loss of a large portion of the β-hemolysin-converting prophage likely contribute to its higher pathogenic potential than the Asian-Pacific clone

A Novel Gene, fudoh, in the SCCmec Region Suppresses the Colony Spreading Ability and Virulence of Staphylococcus aureus

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis

Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered

Human neutrophils phagocytose and kill Acinetobacter baumanii and A. pittii

Acinetobacter baumannii is a common cause of health care associated infections worldwide. A. pittii is an opportunistic pathogen also frequently isolated from Acinetobacter infections other than those from A. baumannii. Knowledge of Acinetobacter virulence factors and their role in pathogenesis is scarce. Also, there are no detailed published reports on the interactions between A. pittii and human phagocytic cells. Using confocal laser and scanning electron microscopy, immunofluorescence, and live-cell imaging, our study shows that immediately after bacteria-cell contact, neutrophils rapidly and continuously engulf and kill bacteria during at least 4 hours of infection in vitro. After 3 h of infection, neutrophils start to release neutrophil extracellular traps (NETs) against Acinetobacter. DNA in NETs colocalizes well with human histone H3 and with the specific neutrophil elastase. We have observed that human neutrophils use large filopodia as cellular tentacles to sense local environment but also to detect and retain bacteria during phagocytosis. Furthermore, co-cultivation of neutrophils with human differentiated macrophages before infections shows that human neutrophils, but not macrophages, are key immune cells to control Acinetobacter. Although macrophages were largely activated by both bacterial species, they lack the phagocytic activity demonstrated by neutrophils

Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA-MRSA pathogenesis are poorly understood and a comprehensive analysis of CA-MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2-dimensional gel electrophoresis coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS) analysis. Eleven known virulence-related exoproteins differed in abundance between the strains, including alpha-haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1-phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host–pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA-MRSA as a human pathogen

Contribution of Panton-Valentine Leukocidin in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains typically carry genes encoding Panton-Valentine leukocidin (PVL). We used wild-type parental and isogenic PVL-deletion (Δpvl) strains of USA300 (LAC and SF8300) and USA400 (MW2) to test whether PVL alters global gene regulatory networks and contributes to pathogenesis of bacteremia, a hallmark feature of invasive staphylococcal disease. Microarray and proteomic analyses revealed that PVL does not alter gene or protein expression, thereby demonstrating that any contribution of PVL to CA-MRSA pathogenesis is not mediated through interference of global gene regulatory networks. Inasmuch as a direct role for PVL in CA-MRSA pathogenesis remains to be determined, we developed a rabbit bacteremia model of CA-MRSA infection to evaluate the effects of PVL. Following experimental infection of rabbits, an animal species whose granulocytes are more sensitive to the effects of PVL compared with the mouse, we found a contribution of PVL to pathogenesis over the time course of bacteremia. At 24 and 48 hours post infection, PVL appears to play a modest, but measurable role in pathogenesis during the early stages of bacteremic seeding of the kidney, the target organ from which bacteria were not cleared. However, the early survival advantage of this USA300 strain conferred by PVL was lost by 72 hours post infection. These data are consistent with the clinical presentation of rapid-onset, fulminant infection that has been associated with PVL-positive CA-MRSA strains. Taken together, our data indicate a modest and transient positive effect of PVL in the acute phase of bacteremia, thereby providing evidence that PVL contributes to CA-MRSA pathogenesis