A novel mouse model of Staphylococcus aureus chronic osteomyelitis that closely mimics the human infection: an integrated view of disease pathogenesis.

Osteomyelitis is a serious bone infection typically caused by Staphylococcus aureus. The pathogenesis of osteomyelitis remains poorly understood, mainly for lack of experimental models that closely mimic human disease. We describe a novel murine model of metastatic chronic osteomyelitis initiated after intravenous inoculation of S. aureus microorganisms. The bacteria entered bones through the bloodstream and, after an acute phase with progressive growth (first 2 weeks after infection), they remained at constant numbers for up to 56 days (chronic phase). Clinical signs of illness and systemic inflammation were apparent only during the acute phase. Bone destruction and remodeling processes were readily detectable by magnetic resonance and X-ray imaging 3 weeks after infection, and high levels of bone deformation were observed during the chronic phase. Histological examination of infected bones demonstrated suppurative inflammation with foci of intense bacterial multiplication and necrosis during acute infection and osteoclastic resorption accompanied by new woven bone formation during chronic infection. Transmission electron microscopy revealed S. aureus microorganisms forming microcolonies within the nonmineralized collagen matrix or located intracellularly within neutrophils. In summary, our mouse model of staphylococcal hematogenous osteomyelitis precisely reproduces most features of the human disease. Although the extent of lesions in the chronic phase was subject to variation, this model is ideal for testing and monitoring novel treatment modalities via noninvasive imaging.

Thiol peroxidase protects Salmonella enterica from hydrogen peroxide stress in vitro and facilitates intracellular growth.

At present, Salmonella is considered to express two peroxiredoxin-type peroxidases, TsaA and AhpC. Here we describe an additional peroxiredoxin, Tpx, in Salmonella enterica and show that a single tpx mutant is susceptible to exogenous hydrogen peroxide (H(2)O(2)), that it has a reduced capacity to degrade H(2)O(2) compared to the ahpCF and tsaA mutants, and that its growth is affected in activated macrophages. These results suggest that Tpx contributes significantly to the sophisticated defense system that the pathogen has evolved to survive oxidative stress.

Identification of a Novel LysR-Type Transcriptional Regulator in Staphylococcus aureus That Is Crucial for Secondary Tissue Colonization during Metastatic Bloodstream Infection.

Staphylococcus aureus is a common cause of bacteremia that can lead to severe complications once the bacteria exit the bloodstream and establish infection in secondary organs. Despite its clinical relevance, little is known about the bacterial factors facilitating the development of these metastatic infections. Here, we used an S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) to identify genes that are critical for efficient bacterial colonization of secondary organs in a murine model of metastatic bloodstream infection. Our transposon screen identified a LysR-type transcriptional regulator (LTTR), which was required for efficient colonization of secondary organs such as the kidneys in infected mice. The critical role of LTTR in secondary organ colonization was confirmed using an isogenic mutant deficient in the expression of LTTR. To identify the set of genes controlled by LTTR, we used an S. aureus strain carrying the LTTR gene in an inducible expression plasmid. Gene expression analysis upon induction of LTTR showed increased transcription of genes involved in branched-chain amino acid biosynthesis, a methionine sulfoxide reductase, and a copper transporter as well as decreased transcription of genes coding for urease and components of pyrimidine nucleotides. Furthermore, we show that transcription of LTTR is repressed by glucose, is induced under microaerobic conditions, and required trace amounts of copper ions. Our data thus pinpoints LTTR as an important element that enables a rapid adaptation of S. aureus to the changing host microenvironment.IMPORTANCEStaphylococcus aureus is an important pathogen that can disseminate via the bloodstream and establish metastatic infections in distant organs. To achieve a better understanding of the bacterial factors facilitating the development of these metastatic infections, we used in this study a Staphylococcus aureus transposon mutant library in a murine model of intravenous infection, where bacteria first colonize the liver as the primary infection site and subsequently progress to secondary sites such as the kidney and bones. We identified a novel LysR-type transcriptional regulator (LTTR), which was specifically required by S. aureus for efficient colonization of secondary organs. We also determined the transcriptional activation as well as the regulon of LTTR, which suggests that this regulator is involved in the metabolic adaptation of S. aureus to the host microenvironment found in secondary infection sites.

Differential Contributions of the Complement Anaphylotoxin Receptors C5aR1 and C5aR2 to the Early Innate Immune Response against Staphylococcus aureus Infection.

The complement anaphylatoxin C5a contributes to host defense against Staphylococcus aureus. In this study, we investigated the functional role of the two known C5a receptors, C5aR1 and C5aR2, in the host response to S. aureus. We found that C5aR1(-/)(-) mice exhibited greater susceptibility to S. aureus bloodstream infection than wild type and C5aR2(-/)(-) mice, as demonstrated by the significantly higher bacterial loads in the kidneys and heart at 24 h of infection, and by the higher levels of inflammatory IL-6 in serum. Histological and immunohistochemistry investigation of infected kidneys at 24 h after bacterial inoculation revealed a discrete infiltration of neutrophils in wild type mice but already well-developed abscesses consisting of bacterial clusters surrounded by a large number of neutrophils in both C5aR1(-/)(-) and C5aR2(-/)(-) mice. Furthermore, blood neutrophils from C5aR1(-/)(-) mice were less efficient than those from wild type or C5aR2(-/)(-) mice at killing S. aureus. The requirement of C5aR1 for efficient killing of S. aureus was also demonstrated in human blood after disrupting C5a-C5aR1 signaling using specific inhibitors. These results demonstrated a role for C5aR1 in S. aureus clearance as well as a role for both C5aR1 and C5aR2 in the orchestration of the inflammatory response during infection.

High-resolution transcriptomic analysis of the adaptive response of Staphylococcus aureus during acute and chronic phases of osteomyelitis.

UNLABELLED: Osteomyelitis is a difficult-to-eradicate bone infection typically caused by Staphylococcus aureus. In this study, we investigated the in vivo transcriptional adaptation of S. aureus during bone infection. To this end, we determined the transcriptome of S. aureus during the acute (day 7) and chronic (day 28) phases of experimental murine osteomyelitis using RNA sequencing (RNA-Seq). We identified a total of 180 genes significantly more highly expressed by S. aureus during acute or chronic in vivo infection than under in vitro growth conditions. These genes encoded proteins involved in gluconeogenesis, proteolysis of host proteins, iron acquisition, evasion of host immune defenses, and stress responses. At the regulatory level, sarA and -R and saeR and -S as well as the small RNA RsaC were predominantly expressed by S. aureus during in vivo infection. Only nine genes, including the genes encoding the arginine deiminase (ADI) pathway and those involved in the stringent response, were significantly more highly expressed by S. aureus during the chronic than the acute stage of infection. Analysis by quantitative reverse transcription-PCR (qRT-PCR) of a subset of these in vivo-expressed genes in clinical specimens yielded the same results as those observed in the murine system. Collectively, our results show that during acute osteomyelitis, S. aureus induced the transcription of genes that mediate metabolic adaptation, immune evasion, and replication. During the chronic phase, however, S. aureus switched its transcriptional response from a proliferative to a persistence mode, probably driven by the severe deficiency in nutrient supplies. Interfering with the survival strategies of S. aureus during chronic infection could lead to more effective treatments. IMPORTANCE: The key to the survival success of pathogens during an infection is their capacity to rapidly adjust to the host environment and to evade the host defenses. Understanding how a pathogen redirects and fine-tunes its gene expression in response to the challenges of infection is central to the development of more efficient anti-infective therapies. Osteomyelitis is a debilitating infection of the bone predominantly caused by S. aureus. In this study, we evaluated the transcriptional response of S. aureus during bone infection. Our results indicate that S. aureus reprograms its genetic repertoire during the acute phase of infection to adapt to nutrient availability and to replicate within the host. During the chronic phase, S. aureus upregulates a survival genetic program activated in response to nutrient starvation. Thus, we have uncovered key survival pathways of S. aureus during acute and chronic osteomyelitis that can be used as therapeutic targets.

Prognostic value and therapeutic potential of TREM-1 in Streptococcus pyogenes- induced sepsis.

TREM-1 (triggering receptor expressed on myeloid cells) is a surface molecule expressed on neutrophils and macrophages which has been implicated in the amplification of inflammatory responses triggered during infection. In the present study, we have investigated the clinical significance of TREM-1 in Streptococcus pyogenes-induced severe sepsis in both experimentally infected mice as well as in patients with streptococcal toxic shock. We found that S. pyogenes induced a dose-dependent upregulation of TREM-1 in in vitro cultured phagocytic cells and in the organs of S. pyogenes-infected mice. Furthermore, we reported a positive correlation between serum levels of soluble TREM-1 (sTREM-1) and disease severity in infected patients as well as in experimentally infected mice. Hence, sTREM-1 may represent a useful surrogate marker for streptococcal sepsis. We found that modulation of TREM-1 by administration of the TREM-1 decoy receptor rTREM-1/Fc substantially attenuated the synthesis of inflammatory cytokines. More importantly, treatment of S. pyogenes-infected septic mice with rTREM-1/Fc or the synthetically produced conserved extracellular domain LP17 significantly improved disease outcome. In summary, our data suggest that TREM-1 may not only represent a valuable marker for S. pyogenes infection severity but it may also be an attractive target for the treatment of streptococcal sepsis.

Methionine sulfoxide reductases are essential for virulence of Salmonella typhimurium.

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H(2)O(2), and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H(2)O(2,) as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium.