CspA regulation of Staphylococcus aureus carotenoid levels and σB activity is controlled by YjbH and Spx.

Staphyloxanthin, a carotenoid in S. aureus, is a powerful antioxidant against oxidative stresses. The crtOPQMN operon driving pigment synthesis is under the control of σB . CspA, a cold shock protein, is known to control σB activity. To ascertain genes that regulate cspA, we screened a transposon library that exhibited reduced cspA expression and pigmentation. We found that the adaptor protein YjbH activates cspA expression. Spx, the redox-sensitive transcriptional regulator and a proteolytic target for YjbH and ClpXP, complexes with αCTD of RNAP prior to binding the cspA promoter to repress cspA activity. Increased cspA expression in trans in the inactive spx C10A mutant of JE2 did not enhance pigment production while it did in JE2, suggesting that cspA is downstream to Spx in pigmentation control. As the staphyloxanthin pigment is critical to S. aureus survival in human hosts, we demonstrated that the cspA and yjbH mutants survived less well than the parent in whole blood killing assay. Collectively, our studies suggest a pathway wherein YjbH and ClpXP proteolytically cleave Spx, a repressor of cspA transcription, to affect σB -dependent carotenoid expression, thus providing a critical link between intracellular redox sensing by Spx and carotenoid production to improve S. aureus survival during infections.

Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection.

Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγnull (NSG) mice engrafted with human CD34+ umbilical cord blood cells. These "humanized" NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL+ versus isogenic PVL- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL+ S. aureus but not PVL- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL+ and PVL- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.

Failure to induce IFN-ß production during Staphylococcus aureus infection contributes to pathogenicity.

The importance of type I IFNs in the host response to viral infection is well established; however, their role in bacterial infection is not fully understood. Several bacteria (both Gram-positive and -negative) have been shown to induce IFN-ß production in myeloid cells, but this IFN-ß is not always beneficial to the host. We examined whether Staphylococcus aureus induces IFN-ß from myeloid phagocytes, and if so, whether it is helpful or harmful to the host to do so. We found that S. aureus poorly induces IFN-ß production compared with other bacteria. S. aureus is highly resistant to degradation in the phagosome because it is resistant to lysozyme. Using a mutant that is more sensitive to lysozyme, we show that phagosomal degradation and release of intracellular ligands is essential for induction of IFN-ß and inflammatory chemokines downstream of IFN-ß. Further, we found that adding exogenous IFN-ß during S. aureus infection (in vitro and in vivo) was protective. Together, the data demonstrate that failure to induce IFN-ß production during S. aureus infection contributes to pathogenicity.

Staphylococcus aureus Panton-Valentine leukocidin targets muscle tissues in a child with myositis and necrotizing fasciitis.

The incidence of myositis has been increasing since the advent of the epidemic of community-associated methicillin-resistant Staphylococcus aureus infection, and Panton-Valentine leukocidin has been implicated as a factor contributing to more-severe muscle injury. We report a case of severe myositis accompanying septic osteomyelitis and necrotizing fasciitis caused by a Panton-Valentine leukocidin-positive S. aureus strain. Immunostaining showed strong binding of the Panton-Valentine leukocidin toxin to necrotic muscle tissues.

Regulation of Rot expression in Staphylococcus aureus.

Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in Staphylococcus aureus. The function of Rot, but not the transcription of rot, is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In addition, the alternative sigma factor (sigma(B)) has a repressive effect on rot expression during the postexponential phase of growth. The transcriptional profiles of Rot in sigma(B)-positive and sigma(B)-negative strains in the postexponential and stationary phases of growth were compared. An upregulation of rot expression was observed during the stationary phase of growth, and this upregulation occurred in a sigma(B)-dependent manner. The effects of other staphylococcal transcriptional factors were also investigated. Electrophoretic mobility shift assays revealed that proteins present in staphylococcal lysates retarded the mobility of the rot promoter fragment and that the effect was reduced, but not eliminated, with lysates from strains lacking a functional SarS protein. A modest upregulation of rot expression was also observed in sarS-negative strains. Affinity purification of proteins binding to the rot promoter fragment, followed by N-terminal protein sequencing, identified the SarA and SarR proteins. Primer extension analysis of the rot promoter revealed a number of discreet products. However, these RNA species were not associated with identifiable promoter activity and likely represented RNA breakdown products. Loss of Rot function during the postexponential phase of growth likely involves degradation of the rot mRNA but not the inhibition of rot transcription.

Group B Streptococcus Evades Host Immunity by Degrading Hyaluronan.

In response to tissue injury, hyaluronan (HA) polymers are cleaved by host hyaluronidases, generating small fragments that ligate Toll-like receptors (TLRs) to elicit inflammatory responses. Pathogenic bacteria such as group B Streptococcus (GBS) express and secrete hyaluronidases as a mechanism for tissue invasion, but it is not known how this activity relates to immune detection of HA. We found that bacterial hyaluronidases secreted by GBS and other Gram-positive pathogens degrade pro-inflammatory HA fragments to their component disaccharides. In addition, HA disaccharides block TLR2/4 signaling elicited by both host-derived HA fragments and other TLR2/4 ligands, including lipopolysaccharide. Application of GBS hyaluronidase or HA disaccharides reduced pulmonary pathology and pro-inflammatory cytokine levels in an acute lung injury model. We conclude that breakdown of host-generated pro-inflammatory HA fragments to disaccharides allows bacterial pathogens to evade immune detection and could be exploited as a strategy to treat inflammatory diseases.

Expanding roles of neutrophils in aging hosts.

Neutrophils' role in the clearance of pathogens is well documented, but there is increasing appreciation that neutrophils can participate in the resolution of infection and inflammation. An obvious implication is that alteration of neutrophil functions with old age could significant impact both susceptibility of the host to infection and inflammatory conditions. Advances in recent years suggest additional chinks in the neutrophil antimicrobial arsenals in aged hosts, which render neutrophils less capable of killing pathogens. Moreover there is evidence that changes in neutrophil cross-talk with other immune cells also contribute to poor resolution of inflammation. These advances provide new insight on how these phagocytic cells could contribute to age-related diseases.

Innate immune dysfunctions in aged mice facilitate the systemic dissemination of methicillin-resistant S. aureus.

Elderly humans show increased susceptibility to invasive staphylococcal disease after skin and soft tissue infection. However, it is not understood how host immunity changes with aging, and how that predisposes to invasive disease. In a model of severe skin infection, we showed that aged mice (16- to 20-month-old) exhibit dramatic bacterial dissemination compared with young adult mice (2-month-old). Bacterial dissemination was associated with significant reductions of CXCL1 (KC), polymorphonuclear cells (PMNs), and extracellular DNA traps (NETs) at the infection site. PMNs and primary skin fibroblasts isolated from aged mice showed decreased secretion of CXCL2 (MIP-2) and KC in response to MRSA, and in vitro analyses of mitochondrial functions revealed that the mitochondrial electron transport chain complex I plays a significant role in induction of chemokines in the cells isolated from young but not old mice. Additionally, PMNs isolated from aged mice have reduced ability to form NETs and to kill MRSA. Expression of nuclease by S. aureus led to increased bacterial systemic dissemination in young but not old mice, suggesting that defective NETs formation in elderly mice permitted nuclease and non-nuclease expressing S. aureus to disseminate equally well. Overall, these findings suggest that gross impairment of both skin barrier function and innate immunity contributes to the propensity for MRSA to disseminate in aged mice. Furthermore, the study indicates that contribution of bacterial factors to pathogenicity may vary with host age.

C/EBPε mediates nicotinamide-enhanced clearance of Staphylococcus aureus in mice.

The myeloid-specific transcription factor, CCAAT/enhancer-binding protein ε (C/EBPε) is a critical mediator of myelopoiesis. Mutation of this gene is responsible for neutrophil-specific granule deficiency in humans, a condition that confers susceptibility to Staphylococcus aureus infection. We found that C/EBPε-deficient mice are severely affected by infection with S. aureus, and C/EBPε deficiency in neutrophils contributes to the infectious phenotype. Conversely, exposure to the epigenetic modulator nicotinamide (vitamin B3) increased expression of C/EBPε in WT myeloid cells. Further, nicotinamide increased the activity of C/EBPε and select downstream antimicrobial targets, particularly in neutrophils. In a systemic murine infection model as well as in murine and human peripheral blood, nicotinamide enhanced killing of S. aureus by up to 1,000 fold but had no effect when administered to either C/EBPε-deficient mice or mice depleted of neutrophils. Nicotinamide was efficacious in both prophylactic and therapeutic settings. Our findings suggest that C/EBPε is an important target to boost killing of bacteria by the innate immune system.

Subcutaneous infection of methicillin resistant Staphylococcus aureus (MRSA).

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) threatens public health worldwide, and epidemiologic data suggest that the Panton-Valentine Leukocidin (PVL) expressed by most CA-MRSA strains could contribute to severe human infections, particularly in young and immunocompetent hosts. PVL is proposed to induce cytolysis or apoptosis of phagocytes. However, recent comparisons of isogenic CA-MRSA strains with or without PVL have revealed no differences in human PMN cytolytic activity. Furthermore, many of the mouse studies performed to date have failed to demonstrate a virulence role for PVL, thereby provoking the question: does PVL have a mechanistic role in human infection? In this report, we evaluated the contribution of PVL to severe skin and soft tissue infection. We generated PVL mutants in CA-MRSA strains isolated from patients with necrotizing fasciitis and used these tools to evaluate the pathogenic role of PVL in vivo. In a model of necrotizing soft tissue infection, we found PVL caused significant damage of muscle but not the skin. Muscle injury was linked to induction of pro-inflammatory chemokines KC, MIP-2, and RANTES, and recruitment of neutrophils. Tissue damage was most prominent in young mice and in those strains of mice that more effectively cleared S. aureus, and was not significant in older mice and mouse strains that had a more limited immune response to the pathogen. PVL mediated injury could be blocked by pretreatment with anti-PVL antibodies. Our data provide new insights into CA-MRSA pathogenesis, epidemiology and therapeutics. PVL could contribute to the increased incidence of myositis in CA-MRSA infection, and the toxin could mediate tissue injury by mechanisms other than direct killing of phagocytes.

Rot repression of enterotoxin B expression in Staphylococcus aureus.

The accessory gene regulator (Agr) system is a quorum-sensing system of Staphylococcus aureus responsible for upregulation of certain exoprotein genes and downregulation of certain cell-wall associated proteins during the post-exponential phase of growth. The enterotoxin B (seb) determinant is upregulated by the Agr system. Agr-regulated cis elements within the seb promoter region were examined by deletion analyses of the seb promoter by a hybrid promoter approach utilizing the staphylococcal lac operon promoter. To identify the regulatory pathway for enterotoxin B expression, the seb promoter fused to the chloramphenicol acetyltransferase reporter gene was introduced into mutants of S. aureus lacking agr or different members of the Sar family of transcriptional regulators. Agr control of seb promoter activity was found to be dependent upon the presence of a functional Rot protein, and Rot was shown to be able to bind to the seb promoter. Therefore, the Agr-mediated post-exponential-phase increase in seb transcription results from the Agr system's inactivation of Rot repressor activity.

Accessory gene regulator control of staphyloccoccal enterotoxin d gene expression.

The quorum-sensing system of Staphylococcus aureus, the accessory gene regulator (Agr) system, is responsible for increased transcription of certain exoprotein genes and decreased transcription of certain cell wall-associated proteins during the postexponential phase of growth. This regulation is important for virulence, as evidenced by a reduction in virulence associated with a loss of the Agr system. The enterotoxin D (sed) determinant is upregulated by the Agr system. To define the Agr-regulated cis element(s) within the sed promoter region, we utilized promoters not regulated by Agr to create hybrid promoters. Hybrid promoters were created by using sed sequences combined with the enterotoxin A (sea) promoter or the S. aureus lac operon promoter sequences. The results obtained indicated that the Agr control element of the sed promoter resides within the -35 promoter element and at the Pribnow box to the +1 site of the promoter. At these positions of the sed promoter, a directly repeated 6-bp sequence was found. This repeat is important for overall promoter activity, and maximal regulation of the promoter activity requires both repeat elements. Furthermore, Agr control of sed promoter activity was found to be dependent upon the presence of a functional Rot protein. Therefore, the postexponential increase in sed transcription results from the Agr-mediated reduction in Rot activity rather than as a direct effect of the Agr system.