Marisa I. Gómez

Staphylococcus aureus activates type I IFN signaling in mice and humans through the Xr repeated sequences of protein A.

The activation of type I IFN signaling is a major component of host defense against viral infection, but it is not typically associated with immune responses to extracellular bacterial pathogens. Using mouse and human airway epithelial cells, we have demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen. This response was dependent on the expression of protein A and, more specifically, the Xr domain, a short sequence-repeat region encoded by DNA that consists of repeated 24-bp sequences that are the basis of an internationally used epidemiological typing scheme. Protein A was endocytosed by airway epithelial cells and subsequently induced IFN-beta expression, JAK-STAT signaling, and IL-6 production. Mice lacking IFN-alpha/beta receptor 1 (IFNAR-deficient mice), which are incapable of responding to type I IFNs, were substantially protected against lethal S. aureus pneumonia compared with wild-type control mice. The profound immunological consequences of IFN-beta signaling, particularly in the lung, may help to explain the conservation of multiple copies of the Xr domain of protein A in S. aureus strains and the importance of protein A as a virulence factor in the pathogenesis of staphylococcal pneumonia.

Regulation of Staphylococcus aureus Capsular Polysaccharide Expression by agr and sarA

ABSTRACT This study addresses the regulation of Staphylococcus aureus type 8 capsular polysaccharide (CP8) expression by the global regulators agr and sarA. We analyzed CP8 production, cap8-specific mRNA synthesis, and blaZ reporter gene activities of the transcriptional and translational fusions in strain Becker and its agr, sarA, and agr-sarA isogenic mutants during different phases of bacterial growth. In the wild-type strain, cap8 mRNA was undetectable until the mid-logarithmic phase of growth, whereas CP8 production was undetectable until 2 h later, at the onset of stationary phase. The delay most likely reflects the time needed for completing CP8 synthesis resulting from translation of cap8 mRNA. The agr mutation caused drastic reductions in CP8 production and cap8 gene transcription, suggesting that agr is a major positive regulator of CP8 expression. The results of gene fusion studies indicated that regulation by agr is exerted at the transcriptional level. In contrast, the sarA mutation caused only a slight reduction in cap8 mRNA synthesis and reporter gene activities. By comparing CP8 production and cap8 transcription, we observed that sarA affected CP8 production both trancriptionally and posttranslationally. We showed that agr was a major activator for cap gene expression not only in type 8 strain Becker but also in strains representing the four agr groups.

Molecular characterization of the capsule locus from non-typeable Staphylococcus aureus

Most Staphylococcus aureus express a serotype 5 or 8 capsular polysaccharide (CP). However, 20–25% of human isolates and up to 86% of bovine strains of S. aureus are non‐typeable (NT), i.e. non‐reactive with antibodies to CP types 1, 2, 5 or 8. A vaccine that targets the S. aureus CP would not protect against NT strains. The aim of this study was to characterize NT S. aureus isolates at the molecular level to explain their lack of type 5 or 8 capsule production. The cap5(8) locus was present in all 22 NT clinical isolates from humans, eight of 21 bovine isolates, and in all eight sequenced strains. NT strains positive for the cap5(8) transcript had mutations within essential capsule genes and could be complemented in trans. S. aureus strains with reduced cap5(8) transcript had mutations within the cap5A promoter, decreased RNAIII levels, or a truncated arlR gene product. More than one mutation was identified in several isolates. The cap5(8) locus was replaced by IS257 in 13 of 21 NT bovine isolates of S. aureus. Lack of capsule expression in NT S. aureus can be explained by multiple mechanisms, and the data argue against the existence of capsule serotypes other than 1, 2, 5 and 8.

Shedding of tumor necrosis factor receptor 1 induced by protein A decreases tumor necrosis factor alpha availability and inflammation during systemic Staphylococcus aureus infection.

ABSTRACT Staphylococcus aureus infections are an important public health concern due to their increasing incidence and high rates of mortality. The success of S. aureus as a pathogen is highly related to its enormous capacity to evade the host immune response. The critical role of tumor necrosis factor alpha (TNF-α) in the initial host defense against systemic staphylococcal infection has been demonstrated in experimental models and may partially explain the lack of significant benefits observed in clinical trials attempting to neutralize this cytokine in septic patients. S. aureus protein A plays a key role in regulating inflammation through its ability to bind and signal through the TNF-α receptor 1 (TNFR1). In this study, we demonstrate that S. aureus, via protein A-mediated signaling, induces early shedding of TNFR1, which precedes the secretion of TNF-α in vitro and in vivo. The results obtained using a protein A-deficient mutant and tnfr1 −/− mice strongly suggest that the increased levels of soluble TNFR1 present during experimental S. aureus infection may neutralize circulating TNF-α and impair the host inflammatory response. Early shedding of TNFR1 induced by protein A may constitute a novel mechanism by which S. aureus subverts the host immune response.

The length of the Staphylococcus aureus protein A polymorphic region regulates inflammation: impact on acute and chronic infection.

Staphylococcus aureus protein A (SpA) plays a critical role in the induction of inflammation. This study was aimed to determine whether the number of short sequence repeats (SSRs) present in the polymorphic region modulates the inflammatory response induced by SpA. We demonstrated that there is a dose-response effect in the activation of interferon (IFN)-β signaling in airway epithelial and immune cells, depending on the number of SSRs, which leads to differences in neutrophil recruitment. We also determined that a significant proportion of isolates from patients with chronic infections such as osteomyelitis and cystic fibrosis carry fewer SSRs than do isolates from patients with acute infections or healthy carriers and that there was an inverse correlation between the number of SSRs and the length of disease course. Given the importance of IFN signaling in eradication of S. aureus, loss of SSRs may represent an advantageous mechanism to adapt to and persist in the host.

The Sbi Protein Contributes to Staphylococcus aureus Inflammatory Response during Systemic Infection.

Staphylococcus aureus is an important human pathogen that causes infections that may present high morbidity and mortality. Among its many virulence factors protein A (SpA) and Staphylococcal binding immunoglobulin protein (Sbi) bind the Fc portion of IgG interfering with opsonophagocytosis. We have previously demonstrated that SpA interacts with the TNF-α receptor (TNFR) 1 through each of the five IgG binding domains and induces the production of pro-inflammatory cytokines and chemokines. The IgG binding domains of Sbi are homologous to those of SpA, which allow us to hypothesize that Sbi might also have a role in the inflammatory response induced by S. aureus. We demonstrate that Sbi is a novel factor that participates in the induction of the inflammatory response during staphylococcal infections via TNFR1 and EGFR mediated signaling as well as downstream MAPKs. The expression of Sbi significantly contributed to IL-6 production and modulated CXCL-1 expression as well as neutrophil recruitment to the site of infection, thus demonstrating for the first time its relevance as a pro-inflammatory staphylococcal antigen in an in vivo model.

Characterization of a New Variant of IS257 That Has Displaced the Capsule Genes within Bovine Isolates of Staphylococcus aureus

ABSTRACT Many bovine Staphylococcus aureus isolates from Argentina are nontypeable (NT), i.e., they do not produce serotype 5 or 8 capsular polysaccharides (CPs). Some of these NT strains have a deletion of the cap5(8) gene cluster mediated by a variant of IS257, now designated IScap. IScap showed 93% amino acid identity to S. aureus ORF49 but only 85% identity to IS431 from S. aureus N315 and 88% identity to an IS257-like element from bovine strain RF122. Thirty-six (53%) of 68 bovine isolates, drawn from a previously described S. aureus strain collection, carried some variant of IS257, including IScap. Of these 36 IS+ isolates, 6 were CP5+, 1 was CP8+, and 29 were NT. Forty-four of the 68 isolates were NT, and 24 of these 44 NT isolates (55%) exhibited IScap-mediated deletion of the cap5(8) gene cluster. IScap was not found among 20 human NT S. aureus isolates bearing the cap5HIJK genes, which suggests that IScap-mediated deletion of the capsule locus is restricted to bovine strains of S. aureus. We were unable to identify a precursor strain in which IScap flanked the cap5(8) capsule locus, nor were we able to select for deletion of the cap5(8) locus in vitro. Our results support the hypothesis that deletion of the cap5 locus occurred in the distant past and that the relative abundance of these NT strains may be a result of their ability to persist in subclinical mastitis infection in cows.

Research articleIntramammary immunization with live-attenuated Staphylococcus aureus: microbiological and immunological studies in a mouse mastitis model

Mammary infection was induced in lactating mice by intramammary injection of Staphylococcus aureus. Histopathological analysis revealed infiltration and lesions of varying magnitude that were still apparent 21 days after the challenge. Concomitantly, viable S. aureus was recovered from infected mammary glands. Mice were immunized by the intramammary route with 5 × 106 colony forming units of a temperature-sensitive mutant of S. aureus and subsequently received a boosting injection seven days later. On day 14 mice were challenged by the intramammary route with the wild-type strain. Intramammary immunization induced a significant increase in milk IgA (P < 0.05), serum IgG (P < 0.05) and serum IgA (P < 0.05) on the day of the challenge, when compared with non-immunized mice. Immunization decreased significantly (P < 0.01) the number of S. aureus colony forming units recovered 96 h after intramammary challenge. In conclusion, the feasibility of immunizing locally with temperature-sensitive S. aureus to induce immunity in the mouse mammary gland was demonstrated. The mouse model of mastitis is proposed as a useful system for screening temperature-sensitive S. aureus strains to be utilized in the development of a vaccine.

Staphylococcus aureus protein A enhances osteoclastogenesis via TNFR1 and EGFR signaling.

Staphylococcus aureus is a major causative agent of osteomyelitis in adults and children. The increasing incidence of antimicrobial resistant isolates and the morbidity of this type of infection denote that alternative therapeutic approaches are required. S. aureus protein A interacts with TNFR1 and EGFR expressed at the surface of host cells. Given the importance of TNF-α and EGFR/RANKL crosstalk in enhancing osteoclast differentiation, the aim of this study was to determine the role of protein A in the induction of osteoclastogenesis and bone resorption during staphylococcal osteomyelitis. We determined that protein A plays a critical role in osteoclast differentiation and activation by initiating TNFR1 and EGFR mediated signaling. Moreover, we demonstrated that protein A significantly contributes to increased osteoclast differentiation and activation as well as cortical bone destruction during the course of disease using experimental models of osteomyelitis. Our findings strongly suggest targeting protein A and TNFR1 as an adjunctive strategy to control bone damage during the initial course of S. aureus osteomyelitis.

Daily very low UV dose exposure enhances adaptive immunity, compared with a single high-dose exposure. Consequences for the control of a skin infection

Ultraviolet radiation (UVr) promotes several well‐known molecular changes, which may ultimately impact on health. Some of these effects are detrimental, like inflammation, carcinogenesis and immunosuppression. On the other hand, UVr also promotes vitamin D synthesis and other beneficial effects. We recently demonstrated that exposure to very low doses of UVr on four consecutive days [repetitive low UVd (rlUVd)] does not promote an inflammatory state, nor the recruitment of neutrophils or lymphocytes, as the exposure to a single high UV dose (shUVd) does. Moreover, rlUVd reinforce the epithelium by increasing antimicrobial peptides transcription and epidermal thickness. The aim of this study was to evaluate the adaptive immune response after shUVd and rlUVd, determining T‐cell and B‐cell responses. Finally, we challenged animals exposed to both irradiation procedures with Staphylococcus aureus to study the overall effects of both innate and adaptive immunity during a cutaneous infection. We observed, as expected, a marked suppression of T‐cell and B‐cell responses after exposure to an shUVd but a novel and significant increase in both specific responses after exposure to rlUVd. However, the control of the cutaneous S. aureus infection was defective in this last group, suggesting that responses against pathogens cannot be ruled out from isolated stimuli.