Real-time monitoring of intracellular Staphylococcus aureus replication

A high-throughput system to rapidly assess the intracellular replication of Staphylococcus aureus has been developed utilizing S. aureus transformed with a dual gfp-luxABCDE reporter operon under the control of a growth-dependent promoter. Replication of tagged bacteria internalized into bovine mammary epithelial cells (MAC-T) could be measured by monitoring fluorescence and bioluminescence from the reporter operon following removal of extracellular bacteria from the plates. Bacterial replication inside cells was confirmed by a novel ex vivo time-lapse confocal microscopic method. This assay of bacterial replication was used to evaluate the efficacy of antibiotics which are commonly used to treat staphylococcal infections. Not all antibiotics tested were able to prevent intracellular replication of S. aureus and some were ineffective at preventing replication of intracellular bacteria at concentrations above the MIC determined for bacteria in broth culture. Comparison of the fluorescence and bioluminescence signals from the bacteria enabled effects on protein synthesis and metabolism to be discriminated and gave information on the entry of compounds into the eukaryotic cell, even if bacterial replication was not prevented. Elevated resistance of S. aureus to antibiotics inside host cells increases the likelihood of selecting S. aureus strains which are resistant to commonly used antimicrobial agents within the intracellular niche. The approach presented directly assesses intracellular efficacy of antibiotics and provides an evidence-based approach to antibiotic selection for prescribing physicians and medical microbiologists.     

Integrin-mediated invasion of Staphylococcus aureus into human cells requires Src family protein-tyrosine kinases

Staphylococcus aureus, a common cause of nosocomial infections, is able to invade eukaryotic cells by indirectly engaging beta1 integrin-containing host receptors, whereas non-pathogenic Staphylococcus carnosus is not invasive. Here, we identify intracellular signals involved in integrin-initiated internalization of S. aureus. In particular, the host cell actin cytoskeleton and Src family protein-tyrosine kinases (PTKs) are essential to mediate S. aureus invasion. Src PTKs are activated in response to pathogenic S. aureus, but not S. carnosus. In addition, pharmacological and genetic interference with Src PTK function reduces bacterial internalization. Importantly, Src PTK-deficient cells are resistant to S. aureus invasion, demonstrating the essentiality of host Src PTKs in integrin-mediated uptake of this pathogen.     

The capacity of bovine endothelial cells to eliminate intracellular Staphylococcus aureus and Staphylococcus epidermidis is increased by the proinflammatory cytokines TNF-alpha and IL-1beta

Staphylococcus aureus is a pathogenic bacterium causing clinical and subclinical bovine mastitis. Infections of the udder by S. aureus are frequently associated with the presence of Staphylococcus epidermidis, an opportunistic pathogen. We reported previously that the capacity of bovine endothelial cells (BEC) to endocytize S. aureus is associated with the activation of NF-kappaB and modulated by the proinflammatory cytokines TNF-alpha and IL-1beta. In this work, we explore the ability of BEC to eliminate intracellular S. aureus and S. epidermidis and their response to these cytokines. Time-kinetics survival experiments indicated that BEC eliminate intracellular S. epidermidis more efficiently. Replication of S. aureus, but not S. epidermidis, inside BEC was evident by an increase in intracellular bacteria recovered at 2 h postinfection. Afterwards, the intracellular number of staphylococci decreased gradually, reaching the lowest value at 24 h. Treatment of BEC with TNF-alpha or IL-1beta potentiated the capacity of BEC to eliminate both Staphylococcus species at the times tested. These results indicate that activation of the intrinsic antistaphylococcal response in BEC, enhanced by TNF-alpha and IL-1beta, is effective to eliminate S. aureus and S. epidermidis and suggest that endothelial cells may play a prominent role in the defense against infections caused by these bacteria.     

Effect of tumor necrosis factor-alpha on intracellular Staphylococcus aureus in vascular endothelial cells.

Although tumor necrosis factor-alpha (TNF-alpha) is an important host factor against intracellular bacteria, little is known about the effect of TNF-alpha on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-alpha influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-alpha, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10(6) CFU well(-1)) into HUVECs for 1 h. TNF-alpha (1 ng mL(-1)) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-alpha plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-alpha preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-alpha helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-alpha preferentially induces apoptosis of endothelial cells infected by S. aureus.     

Effect of tumor necrosis factor-α on intracellular Staphylococcus aureus in vascular endothelial cells

Although tumor necrosis factor-α (TNF-α) is an important host factor against intracellular bacteria, little is known about the effect of TNF-α on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-α influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-α, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10⁶ CFU well⁻¹) into HUVECs for 1 h. TNF-α (1 ng mL⁻¹) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-α plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-α preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-α helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-α preferentially induces apoptosis of endothelial cells infected by S. aureus .     

Apoptosis of human keratinocytes after bacterial invasion

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.     

Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus

Bovine mastitis is a common disease in the dairy industry that causes great economic losses. As the primary pathogen of contagious mastitis, Staphylococcus aureus (S. aureus) can invade bovine mammary epithelial cells, thus evading immune defenses and resulting in persistent infection. Recently, autophagy has been considered an important mechanism for host cells to clear intracellular pathogens. In the current study, autophagy caused by S. aureus was detected, and the correlation between autophagy and intracellular S. aureus survival was assessed. First, a model of intracellular S. aureus infection was established. Then, the autophagy of MAC-T cells was evaluated by confocal microscopy and western blot. Moreover, the activation of the PI3K-Akt-mTOR and ERK1/2 signaling pathways was determined by western blot. Finally, the relationship between intracellular bacteria and autophagy was analyzed by using autophagy regulators (3-methyladenine [3-MA], rapamycin [Rapa] and chloroquine [CQ]). The results showed that S. aureus caused obvious induction of autophagosome formation, transformation of LC3I/II, and degradation of p62/SQSTM1 in MAC-T cells; furthermore, the PI3K-Akt-mTOR and ERK1/2 signaling pathways were activated. The number of intracellular S. aureus increased significantly with autophagy activation by rapamycin, whereas the number decreased when the autophagy flux was inhibited by chloroquine. Therefore, this study indicated that intracellular S. aureus can induce autophagy and utilize it to survive in bovine mammary epithelial cells.     

IFN-gamma regulated chemokine production determines the outcome of Staphylococcus aureus infection

Immunomodulatory therapy represents an attractive approach in treating multidrug-resistant infections. Developing this therapy necessitates a lucid understanding of host defense mechanisms. Neutrophils represent the first line of systemic defense during Staphylococcus aureus infections. However, recent research suggests that survival of S. aureus inside neutrophils may actually contribute to pathogenesis, indicating that neutrophil trafficking to the infection site must be tightly regulated to ensure efficient microbial clearance. We demonstrate that neutrophil-regulating T cells are activated during S. aureus infection and produce cytokines that control the local neutrophil response. S. aureus capsular polysaccharide activates T cell production of IFN-gamma in a novel MHC class II-dependent mechanism. During S. aureus surgical wound infection, the presence of IFN-gamma at the infection site depends upon alphabetaTCR+ cells and functions to regulate CXC chemokine production and neutrophil recruitment in vivo. We note that the reduced neutrophil response seen in IFN-gamma-/- mice during S. aureus infection is associated with reduced tissue bacterial burden. CXC chemokine administration to the infection site resulted in an increased survival of viable S. aureus inside neutrophils isolated from the wound. These data demonstrate that T cell-derived IFN-gamma generates a neutrophil-rich environment that can potentiate S. aureus pathogenesis by facilitating bacterial survival within the neutrophil. These findings suggest avenues for novel immunomodulatory approaches to control S. aureus infections.     

Factors influencing the internalization of Staphylococcus aureus and impacts on the course of infections in humans

Staphylococcus aureus is the primary etiological agent of several human diseases. S. aureus has classically been considered an extracellular pathogen; however, recent evidence indicates that S. aureus invades and persists in non-professional phagocytes. Experiments demonstrate that actin microfilaments, microtubules, receptor-mediated endocytosis, and protein tyrosine kinases play important roles in the uptake of S. aureus. Fibronectin-binding proteins and beta-integrins are implicated as critical cell surface molecules associated with internalization of S. aureus by non-phagocytic cells. Following invasion of eukaryotic cells, S. aureus induces the release of cytokines that have the potential to exacerbate disease and induce apoptosis. Finally, S. aureus has the ability to persist inside host cells as small colony variants, a phenotype associated with persistent and recurrent infections.     

Molecular docking analysis of Enterotoxin I from Staphylococcus aureus with Nafcillin analogues

Staphylococcal enterotoxins (SEs) are liked with food poisoning and other related infections. Nafcillin is an antibiotic used to treat S. aureus. Therefore, it is of interest to study the molecular interactions of 25 nafcillin analogues with enterotoxin I using molecular docking analysis. The analysis shows optimal interaction features of Nafcillin analogues with Enterotoxin I from Staphylococcus aureus for further consideration.     

TRAIL expression is induced in both osteoblasts containing intracellular Staphylococcus aureus and uninfected osteoblasts in infected cultures

Staphylococcus aureus is the principal etiological agent of osteomyelitis (bone infection), which is characterized by a progressive inflammatory response resulting in extensive damage to bone tissue. Recent studies have demonstrated the ability of S. aureus to invade and persist inside osteoblasts (bone matrix-forming cells) and other eukaryotic cells. The presence of intracellular S. aureus in bone tissue may be relevant to the pathology of osteomyelitis, a disease often refractory to antibiotic treatment and subject to recurrence months and even years after apparently successful therapy. The present study examined the production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following S. aureus infection, and whether expression of the molecule was induced by those osteoblasts containing intracellular S. aureus. Results from this study suggest that osteoblasts containing intracellular S. aureus induce TRAIL expression in uninfected osteoblasts present in infected cultures.     

Survival of Staphylococcus aureus inside neutrophils contributes to infection

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar-) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.     

Conspicuous ingestion of Staphylococcus aureus organisms by murine fibroblasts in vitro.

A conspicuous adhesion of Staphylococcus aureus organisms to murine cutaneous fibroblasts and NIH/3T3 cells cultured in vitro and subsequent ingestion of S. aureus organisms by these fibroblasts are described. In the present experimental system, only fibroblasts-adhering S. aureus organisms were efficiently ingested by fibroblasts unlike S. epidermidis and S. saprophyticus. These findings might suggest a correlation between the pathogenesis of S. aureus and its intracellular localization in non-professional phagocytes such as fibroblasts in a special reference to its higher pathogenicity than those of coagulase negative counterparts.     

Phevalin (aureusimine B)production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression

Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.     

Anti-infective properties of Lactobacillus fermentum against Staphylococcus aureus and Pseudomonas aeruginosa

Surgical wounds and implant-associated Staphylococcus aureus and Pseudomonas aeruginosa infections are often difficult to treat because of limited susceptibility of several of these strains to conventional antibiotics. As a result, there is a constant need for new alternative drugs. The aim of this study was to investigate the antimicrobial properties of Lactobacillus fermentum, a probiotic bacterium, which we have isolated from colonic biopsies. The inhibition of S. aureus and P. aeruginosa growth was evaluated by coincubating with L. fermentum strains. Growth inhibition was tested for several of their clinical isolates using agar well diffusion assays. For biofilm assay S. aureus and P. aeruginosa were grown on the glass slides and in 96-well plates in presence of 2.5 μg/ml culture filtrate of L. fermentum. Biofilms were photographed using confocal microscope or stained with 0.1% crystal violet. Reduction in the cytotoxicity of S. aureus and P. aeruginosa was observed in presence of 2.5 μg/ml L. fermentum-spent media. Using in vitroexperiments, we showed that L. fermentum-secreted compound(s) inhibits the growth, cytotoxicity and biofilm formation of several S. aureus and P. aeruginosa strains. Compound(s) present in the culture supernatant of L. fermentum may have promising applications in treating hospital-acquired infections.     

Invasion of bone cells by Staphylococcus epidermidis

Bone implants infected with Staphylococcus epidermidis often require surgical intervention because of the failure of antibiotic treatment. The reasons why such infections are resistant to therapy are poorly understood. We have previously reported that another bacterium, Staphylococcus aureus, can invade bone cells and thereby evade antimicrobial therapy. In this study we have investigated the hypothesis that S. epidermidis can also invade bone cells and may therefore explain the difficulties of treating infections with this organism. We found that S. epidermidis was capable of invading bone cells but that there were significant strain dependent differences in this capacity. A recombinant protein encompassing the D1-D4 repeat region of S. aureus fibronectin-binding protein B completely inhibited internalization of S. aureus but failed to block internalization of S. epidermidis. Similarly a blocking antibody to alpha5beta1 integrin inhibited internalization of S. aureus by bone cells but had no effect on the uptake of S. epidermidis. Therefore unlike S. aureus, S. epidermidis does not gain entrance into bone cells through a fibronectin bridge between the alpha5beta1 integrin and a bacterial adhesin.     

Multiple virulence factors are required for Staphylococcus aureus-induced apoptosis in endothelial cells

Staphylococcus aureus infections can result in sepsis and septic shock associated with vascular damage and multiple organ failure. Apoptosis appears to play a key role during sepsis, and the ability of S. aureus to induce apoptosis in endothelial cells might contribute to metastatic infection. In contrast to leukocytes, in human umbilical vein endothelial cells and two endothelial cell lines neither purified alpha-toxin nor staphylococcal supernatants were sufficient to induce apoptosis. Apoptosis induction instead required staphylococcal invasion as well as signals from metabolically active intracellular staphylococci. Only strongly haemolytic and invasive staphylococci, but not non-invasive strains induced apoptosis that was caspase-dependent but Fas-independent. However, only a subgroup of clinical isolates with an invasive and haemolytic phenotype induced apoptosis. Expression of alpha-toxin in a non-haemolytic strain partially restored apoptosis induction, suggesting a role of alpha-toxin as a trigger of apoptosis. Furthermore, infection of endothelial cells with isogenic mutants of various regulator genes revealed that apoptosis induction was dependent on the global regulator agr and the alternative sigma factor sigB, but not influenced by sarA. Together, our results indicate that the ability of S. aureus to induce apoptosis in endothelial cells is determined by multiple virulence factors.