Lorena Tuchscherr

Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection

Staphylococcus aureus is a frequent cause for serious, chronic and therapy‐refractive infections in spite of susceptibility to antibiotics in vitro. In chronic infections, altered bacterial phenotypes, such as small colony variants (SCVs), have been found. Yet, it is largely unclear whether the ability to interconvert from the wild‐type to the SCV phenotype is only a rare clinical and/or just laboratory phenomenon or is essential to sustain an infection. Here, we performed different long‐term in vitro and in vivo infection models with S. aureus and we show that viable bacteria can persist within host cells and/or tissues for several weeks. Persistence induced bacterial phenotypic diversity, including SCV phenotypes, accompanied by changes in virulence factor expression and auxotrophism. However, the recovered SCV phenotypes were highly dynamic and rapidly reverted to the fully virulent wild‐type form when leaving the intracellular location and infecting new cells. Our findings demonstrate that bacterial phenotype switching is an integral part of the infection process that enables the bacteria to hide inside host cells, which can be a reservoir for chronic and therapy‐refractive infections.

Staphylococcus aureus Small-Colony Variants Are Adapted Phenotypes for Intracellular Persistence

BACKGROUND Staphylococcus aureus is an important human pathogen of endovascular diseases, which can take a chronic course with a high relapse rate despite antimicrobial treatment. Thus far, persistent and antibiotic-refractory infections have been largely associated with a subpopulation of S. aureus, the small-colony variants (SCVs). METHODS In this work, we used endothelial cells to investigate infection with the highly virulent wild-type isolate (6850), 2 stable isogenic SCV phenotypes (hemB mutant IIb13 and JB1), and the complemented mutant. RESULTS All strains were highly invasive in endothelial cells but largely differed in host response induction. Microarray analysis showed that wild-type phenotypes up-regulated a large number of endothelial genes (including genes involved in innate immunity), whereas the SCVs did not cause these dramatic changes. The inflammatory response and cytotoxicity were strongest shortly after infection and largely decreased within the following days, which was accompanied by a fast elimination of intracellular wild-type bacteria. By contrast, SCVs survived within endothelial cells at high numbers. CONCLUSION S. aureus intracellular persistence via the development of an adapted subpopulation of SCVs most likely represents an important strategy of S. aureus to hide within the host cells, which could be a reservoir for chronic infections.

Antibodies to Capsular Polysaccharide and Clumping Factor A Prevent Mastitis and the Emergence of Unencapsulated and Small-Colony Variants of Staphylococcus aureus in Mice

ABSTRACT The pathogenesis of Staphylococcus aureus infections is influenced by multiple virulence factors that are expressed under variable conditions, and this has complicated the design of an effective vaccine. Clinical trials that targeted the capsule or clumping factor A (ClfA) failed to protect the recipients against staphylococcal infections. We passively immunized lactating mice with rabbit antibodies to S. aureus capsular polysaccharide (CP) serotype 5 (CP5) or CP8 or with monoclonal antibodies to ClfA. Mice immunized with antibodies to CP5 or CP8 or with ClfA had significantly reduced tissue bacterial burdens 4 days after intramammary challenge with encapsulated S. aureus strains. After several passages in mice passively immunized with CP-specific antiserum, increasing numbers of stable unencapsulated variants of S. aureus were cultured from the infected mammary glands. Greater numbers of these unencapsulated S. aureus variants than of the corresponding encapsulated parental strains were internalized in vitro in MAC-T bovine cells. Furthermore, small-colony variants (SCVs) were recovered from the infected mammary glands after several passages in mice passively immunized with CP-specific antiserum. A combination of antibodies effectively sterilized mammary glands in a significant number of passively immunized mice. More importantly, passive immunization with antibodies to both CP and ClfA fully inhibited the emergence of unencapsulated “escape mutants” and significantly reduced the appearance of SCVs. A vaccine formulation comprising CP conjugates plus a surface-associated protein adhesin may be more effective than either antigen alone for prevention of S. aureus infections.

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.

Differential Abilities of Capsulated and Noncapsulated Staphylococcus aureus Isolates from Diverse agr Groups To Invade Mammary Epithelial Cells

ABSTRACT Staphylococcus aureus is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (agr) system. The aim of this study was to ascertain the prevalence of the different agr groups in capsulated and noncapsulated S. aureus bacteria isolated from mastitic bovines in Argentina and whether a given agr group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine S. aureus strains were classified in agr group I. The remainder belonged in agr groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the agr locus variable region, six agr restriction types were identified. All agr group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine S. aureus strains defined as being in agr group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from agr groups II, III, and IV. agr group II or IV S. aureus strains were cleared more efficiently than agr group I strains from the murine mammary gland. The results suggest that agr group I S. aureus strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than S. aureus strains classified in agr group II, III, or IV.

Staphylococcal Strains Vary Greatly in Their Ability to Induce an Inflammatory Response in Endothelial Cells

BACKGROUND Staphylococcus aureus is an important human pathogen of endovascular diseases that can take an acute destructive course and/or develop into a chronic infection with a high rate of relapse. Despite good activity of the appropriate antimicrobial substances in vitro, these infections are often difficult to treat, perhaps because of the complex infection process of endovascular tissue. METHODS We used the microarray technique to analyze the response of endothelial cells to different S. aureus strains, including highly virulent isolates (6850 and ST239); the Cowan I strain, which has a nonfunctioning accessory gene regulator (agr) quorum-sensing system; and various clinical isolates. RESULTS All tested strains were equally invasive in endothelial cells and were found intracellularly, but they differed greatly in their ability to induce inflammation. Wild-type isolates (6850 and ST239) up-regulated a huge number of genes, including many genes involved in innate immunity. By contrast, Cowan I, which failed to express important virulence factors related to the agr system (eg, alpha-toxin and proteases), did not induce these dramatic changes in endothelial gene expression. Similar results were obtained with clinical isolates. CONCLUSION Given that agr-defective strains are commonly recovered during colonization and infection, agr deficiency might represent a strategy of S. aureus to hide intracellularly without provoking the host immune system and causing relapsing infections.

Staphylococcus aureus persistence in non-professional phagocytes

S. aureus is a frequent cause of chronic and therapy-refractory infections. The ability of S. aureus to invade different types of non-professional phagocytes, to escape from the host lysosomal degradation machinery and to persist within the intracellular location for long time periods are most likely essential steps in pathogenesis. During the course from acute to chronic infection the bacteria need to dynamically react to the environmental changes and to adapt to the intracellular environment. In this context the bacteria change to SCV-like phenotypes that exhibit some characteristics of stable SCV-mutants, like upregulation of adhesins and downregulation of toxins. The exact formation mechanism and further typical features of these dynamically forming SCVs are largely unknown. In this review, recent data on the essential steps to establish chronic infections will be summarized and the clinical consequences of the dynamic bacterial adaptation mechanisms will be discussed.

Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections

Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.

Combined Action of Influenza Virus and Staphylococcus aureus Panton–Valentine Leukocidin Provokes Severe Lung Epithelium Damage

Staphylococcus aureus necrotizing pneumonia is a life-threatening disease that is frequently preceded by influenza infection. The S. aureus toxin Panton–Valentine leukocidin (PVL) is most likely causative for necrotizing diseases, but the precise pathogenic mechanisms of PVL and a possible contribution of influenza virus remain to be elucidated. In this study, we present a model that explains how influenza virus and PVL act together to cause necrotizing pneumonia: an influenza infection activates the lung epithelium to produce chemoattractants for neutrophils. Upon superinfection with PVL-expressing S. aureus, the recruited neutrophils are rapidly killed by PVL, resulting in uncontrolled release of neutrophil proteases that damage the airway epithelium. The host counteracts this pathogen strategy by generating PVL-neutralizing antibodies and by neutralizing the released proteases via protease inhibitors present in the serum. These findings explain why necrotizing infections mainly develop in serum-free spaces (eg, pulmonary alveoli) and open options for new therapeutic approaches.

Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice.

ABSTRACT Staphylococcus aureus capsular polysaccharides (CP) have been shown to enhance staphylococcal virulence in numerous animal models of infection. Although serotype 5 CP (CP5) and CP8 predominate among S. aureus isolates from humans, most staphylococcal isolates from bovines with mastitis in Argentina are capsule negative. This study was designed to evaluate the effects of CP5 and CP8 expression on the pathogenesis of experimental murine mastitis. Lactating mice were challenged by the intramammary route with one of three isogenic S. aureus strains producing CP5, CP8, or no capsule. Significantly greater numbers of acapsular mutant cells were recovered from the infected glands 12 days after bacterial challenge compared with the encapsulated strains. Histopathological analyses revealed greater polymorphonuclear and mononuclear leukocyte infiltration and congestion in the mammary glands of mice infected with the encapsulated strains compared with the acapsular mutant, and the serotype 5 strain elicited more inflammation than the serotype 8 strain. In vitro experiments revealed that the acapsular S. aureus strain was internalized by MAC-T bovine epithelial cells in significantly greater numbers than the CP5- or CP8-producing strain. Taken together, the results suggest that S. aureus lacking a capsule was able to persist in the murine mammary gland, whereas encapsulated strains elicited more inflammation and were eliminated faster. Loss of CP5 or CP8 expression may enhance the persistence of staphylococci in the mammary glands of chronically infected hosts.