Cytoplasmic replication of Staphylococcus aureus upon phagosomal escape triggered by phenol‐soluble modulin α

Staphylococcus aureus is a Gram‐positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non‐professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S. aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol‐soluble modulins (PSM) have been identified to comprise a genus‐specific family of cytolytic peptides. Of these the PSMα peptides have been implicated in killing polymorphonuclear leucocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin‐resistant S. aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin‐sensitive strain 6850 were compared with their respective wild type strains. In all three genetic backgrounds, PSMα mutants were unable to escape from phagosomes in non‐professional (293, HeLa, EAhy.926) and professional phagocytes (THP‐1), whereas mutants in PSMβ and δ‐toxin as well as β‐toxin, phosphatidyl inositol‐dependent phospholipase C and Panton Valentine leucotoxin escaped with efficiencies of the parental strains. S. aureus replicated intracellularly only in presence of a functional PSMα operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape.     

Influence of Sae‐regulated and Agr‐regulated factors on the escape of Staphylococcus aureus from human macrophages

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild‐type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol‐soluble modulins (PSMs) for phagosomal escape. Thus, Sae‐regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis‐related caspase 3, 7 or 8 nor to NLRP3‐dependent inflammasome activation.     

Inability to sustain intraphagolysosomal killing of Staphylococcus aureus predisposes to bacterial persistence in macrophages

Macrophages are critical effectors of the early innate response to bacteria in tissues. Phagocytosis and killing of bacteria are interrelated functions essential for bacterial clearance but the rate‐limiting step when macrophages are challenged with large numbers of the major medical pathogen Staphylococcus aureus is unknown. We show that macrophages have a finite capacity for intracellular killing and fail to match sustained phagocytosis with sustained microbial killing when exposed to large inocula of S. aureus (Newman, SH1000 and USA300 strains). S. aureus ingestion by macrophages is associated with a rapid decline in bacterial viability immediately after phagocytosis. However, not all bacteria are killed in the phagolysosome, and we demonstrate reduced acidification of the phagolysosome, associated with failure of phagolysosomal maturation and reduced activation of cathepsin D. This results in accumulation of viable intracellular bacteria in macrophages. We show macrophages fail to engage apoptosis‐associated bacterial killing. Ultittop mately macrophages with viable bacteria undergo cell lysis, and viable bacteria are released and can be internalized by other macrophages. We show that cycles of lysis and reuptake maintain a pool of viable intracellular bacteria over time when killing is overwhelmed and demonstrate intracellular persistence in alveolar macrophages in the lungs in a murine model.     

Microbiome precision editing: Using PEG as a selective fermentation initiator against methicillin‐resistant Staphylococcus aureus

Recent creation of a Unified Microbiome Initiative (UMI) has the aim of understanding how microbes interact with each other and with us. When pathogenic Staphylococcus aureus infects the skin, the interplay between S. aureus and skin commensal bacteria occurs. Our previous data revealed that skin commensal bacteria can mediate fermentation against the growth of USA300, a community‐acquired methicillin‐resistant S. aureus MRSA. By using a fermentation process with solid media on a small scale, we define poly(ethylene glycol) dimethacrylate (PEG‐DMA) as a selective fermentation initiator which can specifically intensify the probiotic ability of skin commensal Staphylococcus epidermidis bacteria. At least five short‐chain fatty acids including acetic, butyric and propionic acids with anti‐USA300 activities are produced by PEG‐DMA fermentation of S. epidermidis. Furthermore, the S. epidermidis‐laden PEG‐DMA hydrogels effectively decolonized USA300 in skin wounds in mice. The PEG‐DMA and its derivatives may become novel biomaterials to specifically tailor the human skin microbiome against invading pathogens.     

Shedding light on host niches: label‐free in situ detection of Mycobacterium gordonae via carotenoids in macrophages by Raman microspectroscopy

Macrophages are the primary habitat of pathogenic mycobacteria during infections. Current research about the host–pathogen interaction on the cellular level is still going on. The present study proves the potential of Raman microspectroscopy as a label‐free and non‐invasive method to investigate intracellular mycobacteria in situ. Therefore, macrophages were infected with Mycobacterium gordonae, a mycobacterium known to cause inflammation linked to intracellular survival in macrophages. Here, we show that Raman maps provided spatial and spectral information about the position of bacteria within determined cell margins of macrophages in two‐dimensional scans and in three‐dimensional image stacks. Simultaneously, the relative intracellular concentration and distributions of cellular constituents such as DNA, proteins and lipids provided phenotypic information about the infected macrophages. Locations of bacteria outside or close to the outer membrane of the macrophages were notably different in their spectral pattern compared with intracellular once. Furthermore, accumulations of bacteria inside of macrophages exhibit distinct spectral/molecular information because of the chemical composition of the intracellular microenvironment. The data show that the connection of microscopically and chemically gained information provided by Raman microspectroscopy offers a new analytical way to detect and to characterize the mycobacterial infection of macrophages.     

A new host cell internalisation pathway for SadA‐expressing staphylococci triggered by excreted neurochemicals

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface‐bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal‐pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface‐bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT‐29. We found that TAs and DOP are agonists of the α2‐adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F‐actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F‐actin formation without decreasing the cytoplasmic cAMP level. The neurochemical‐induced internalisation in host cells is independent of the fibronectin‐binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.     

A privileged intraphagocyte niche is responsible for disseminated infection of Staphylococcus aureus in a zebrafish model

The innate immune system is the primary defence against the versatile pathogen, Staphylococcus aureus. How this organism is able to avoid immune killing and cause infections is poorly understood. Using an established larval zebrafish infection model, we have shown that overwhelming infection is due to subversion of phagocytes by staphylococci, allowing bacteria to evade killing and found foci of disease. Larval zebrafish coinfected with two S. aureus strains carrying different fluorescent reporter gene fusions (but otherwise isogenic) had bacterial lesions, at the time of host death, containing predominantly one strain. Quantitative data using two marked strains revealed that the strain ratios, during overwhelming infection, were often skewed towards the extremes, with one strain predominating. Infection with passaged bacterial clones revealed the phenomenon not to bedue to adventitious mutations acquired by the pathogen. After infection of the host, all bacteria are internalized by phagocytes and the skewing of population ratios is absolutely dependent on the presence of phagocytes. Mathematical modelling of pathogen population dynamics revealed the data patterns are consistent with the hypothesis that a small number of infected phagocytes serve as an intracellular reservoir for S. aureus, which upon release leads to disseminated infection. Strategies to specifically alter neutrophil/macrophage numbers were used to map the potential subpopulation of phagocytes acting as a pathogen reservoir, revealing neutrophils as the likely ‘niche’. Subsequently in a murine sepsis model, S. aureus abscesses in kidneys were also found to be predominantly clonal, therefore likely founded by an individual cell, suggesting a potential mechanism analogous to the zebrafish model with few protected niches. These findings add credence to the argument that S. aureus control regimes should recognize both the intracellular as well as extracellular facets of the S. aureus life cycle.     

Staphylococcal alpha‐phenol soluble modulins contribute to neutrophil lysis after phagocytosis

Staphylococcus aureus community‐acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA‐MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA‐MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α‐type and β‐type PSMs on neutrophil killing after phagocytosis. Using fluorescently labelled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time‐lapse microscopy. Phagocytosis of the CA‐MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increasedneutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.     

BigA is a novel adhesin of Brucella that mediates adhesion to epithelial cells

Adhesion to cells is the initial step in the infectious cycle of basically all pathogenic bacteria, and to do so, microorganisms have evolved surface molecules that target different cellular receptors. Brucella is an intracellular pathogen that infects a wide range of mammals whose virulence is completely dependent on the capacity to replicate in phagocytes. Although much has been done to elucidate how Brucella multiplies in macrophages, we still do not understand how bacteria invade epithelial cells to perform a replicative cycle or what adhesion molecules are involved in the process. We report the identification in Brucella abortus of a novel adhesin that harbours a bacterial immunoglobulin‐like domain and demonstrate that this protein is involved in the adhesion to polarized epithelial cells such as the Caco‐2 and Madin–Darby canine kidney models targeting the bacteria to the cell–cell interaction membrane. While deletion of the gene significantly reduced adhesion, over‐expression dramatically increased it. Addition of the recombinant protein to cells induced cytoskeleton rearrangements and showed that this adhesin targets proteins of the cell–cell interaction membrane in confluent cultures.     

Plume height and surface coverage analysis of methicillin-resistant Staphylococcus aureus isolates grown in a CDC biofilm reactor

Biofilm formation is a dynamic process that leads to mature communities over time. Despite a general knowledge of biofilm community formation and the resultant limitations of antibiotic therapy, there is a paucity of data describing specific plume heights, surface coverage and rates of maturation. Furthermore, little is published on the effect that the broth medium might have on the degree of biofilm maturation. In this study, three strains of methicillin-resistant Staphylococcus aureus (MRSA) (USA300, USA400 and a clinical isolate) were grown in brain heart infusion broth (BHI) or tryptic soy broth (TSB). Following growth, SEM images were captured for 3-D analysis to assess plume height. TSB produced significantly higher plume heights of USA300 and USA400 compared to BHI. Broth type was less influential on the clinical isolate. The data indicate that broth type and time may be important factors to consider when assessing maturation and plume height formation of MRSA biofilms.     

Piscirickettsia salmonis induces apoptosis in macrophages and monocyte‐like cells from rainbow trout

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS) which causes significant losses in salmon production in Chile and other and in other regions in the southern hemisphere. As the killing of phagocytes is an important pathogenic mechanism for other bacteria to establish infections in vertebrates, we investigated whether P. salmonis kills trout macrophages by apoptosis. Apoptosis in infected macrophages was demonstrated by techniques based on morphological changes and host cell DNA fragmentation. Transmission electron microcopy showed classic apoptotic characteristics and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling showed fragmented DNA. Programmed cell death type I was further confirmed by increased binding of annexin V to externalized phosphatidylserine in infected macrophages. Moreover, significant increases of caspase 3 activation were detected in infected cells and treatment with caspase inhibitor caused a decrease in levels of apoptosis. This is the first evidence that P. salmonis induces cell death in trout macrophages. This could lead to bacterial survival and evasion of the host immune response and play an important role in the establishment of infection in the host. J. Cell. Biochem. 110: 468–476, 2010. © 2010 Wiley‐Liss, Inc.     

Autophagosomes can support Yersinia pseudotuberculosis replication in macrophages

Yersinia pseudotuberculosis is able to replicate inside macrophages. However, the intracellular trafficking of the pathogen after its entry into the macrophage remains poorly understood. Using in vitro infected bone marrow‐derived macrophages, we show that Y. pseudotuberculosis activates the autophagy pathway. Host cell autophagosomes subverted by bacteria do not become acidified and sustain bacteria replication. Moreover, we report that autophagy inhibition correlated with bacterial trafficking inside an acidic compartment. This study indicates that Y. pseudotuberculosis hijacks the autophagy pathway for its replication and also opens up new opportunities for deciphering the molecular basis of the host cell signalling response to intracellular Yersinia infection.     

Experimental Salmonellosis

When mononuclear phagocytes (macrophages), were infected with Salmonella enteritidis and confined in a diffusion chamber to incubate with normal macrophages, they confer on the normal macrophages cellular immunity, as detected by inhibition to intracellular multiplication of a virulent strain 116-54 and resistance to cellular degeneration caused by phagocytosis of bacteria. An immune ribonucleic acid (RNA) was extracted from the peritoneal macrophages maintained in tissue culture bottles in a homogeneous cell population which had been infected with strains of 5. enteritidis. When peritoneal macrophages, cultured in a homogeneous cell population, were treated in vitro with this agent, they developed cellular immunity and cellular antibody. The RNA preparation was not inactived by treatment with deoxyribonuclease, with pronase or with antibodies to a virulent strain 116-54 of S. enteritidis. These facts suggest that the macrophages constitute a cell line responsible for active antibody formation.     

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin‐derived protein

New anti‐infective approaches are much needed to control multi‐drug‐resistant (MDR) pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4‐ to 23.4‐fold as response to V12CBD. More significantly, V12CBD could activate macrophages through NF‐κB pathway and enhance phagocytosis against S. aureus. As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections.     

Enumeration of phagocytosed Staphylococcus aureus inside cells of the mouse macrophage cell line J774 by bioluminescence,fluorescence and viable counting techniques

In order to quantify intracellular Staphylococcus aureus within a macrophage-like cell line by a bioluminescence technique, the mouse cell line J774 and opsonized Staphylococcus aureus were incubated together to allow phagocytosis to occur. Experiments using UV microscopy and fluorescent stained S. aureus were performed to determine an estimate of the mean intracellular bacterial numbers. For enumeration of intracellular bacteria by a bioluminescence technique, extracellular bacteria were removed by washing, the macrophages lysed mechanically and osmotically and treated with apyrase to remove somatic ATP. Bacterial cells were washed and the intracellular ATP measured by firefly luciferase bioluminescence in a luminometer. This new method of enumerating intracellular bacteria was compared to the conventional method of viable counts and found to correlate (r = 0.78). The bioluminescence assay developed was found to be a relatively rapid alternative method to the techniques currently used to enumerate intracellular bacteria and could prove advantageous in studies of intracellular killing and effects of antimicrobial agents on intracellular pathogens.     

Characterization of Methicillin-resistant Staphylococcus aureus isolated from public surfaces on a university campus, student homes and local community

Aim: Isolation and characterization of methicillin‐resistant Staphylococcus aureus (MRSA) from frequently touched nonhospital environmental surfaces at a large university, student homes and community sites. Methods and Results: Twenty‐four isolates from 21 (4·1%, n = 509) surfaces were MRSA positive and included 14 (58%, n = 24) SCCmec type IV, two (8%, n = 24) type I, and eight (33%, n = 24) were not type I‐IV (NT). Six different multilocus sequencing types were identified by PCR and sequencing. PCR assays identified one (4·2%, n = 24) Panton‐Valentine leukocidin (PVL) positive, 22 (92%, n = 24) arginine catabolic mobile element (ACME) positive and 23 (96%, n = 24) multidrug‐resistant (kanamycin, macrolide, tetracycline) MRSA isolates. Eleven (46%, n = 24) USA300 isolates were determined by pulsed‐field gel electrophoresis. Conclusion: The MRSA‐positive environmental surfaces were identified in student homes (11·8%, n = 85), the community (2·3%, n = 130) and the university (2·7%, n = 294). USA300 strains were isolated from the university, student homes and community samples. This is the first report of the animal clone ST97 on urban environmental surfaces. Significance and Impact of the Study: The study highlights the distribution of USA300 on frequently touched surfaces. Whether contact with these MRSA contaminated environmental surfaces are associated with increased risk of transmission of MRSA to people needs further research.     

The Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 replicates in mature phagolysosomes within J774 macrophages

Adherent-invasive Escherichia coli (AIEC) bacteria isolated from Crohn's disease patients are able to extensively replicate within macrophages in large vacuoles. The mechanism by which AIEC bacteria survive within phagocytic cells is unknown. This report describes the maturation of AIEC LF82-containing phagosomes within J774 macrophages. LF82-containing phagosomes traffic through the endocytic pathway as shown by the sequential acquisition and loss of EEA1 and Rab7 and by accumulation of Lamp-1, Lamp-2 and cathepsin D. We demonstrated that AIEC LF82-containing phagosomes mature into active phagolysosomes where bacteria are exposed to low pH and to the degradative activity of cathepsin D. Finally, we showed that an acidic environment is necessary for replication of AIEC LF82 bacteria within J774 macrophages. Thus, evidence is provided that AIEC LF82 bacteria do not escape from the endocytic pathway but undergo normal interaction with host endomembrane organelles and replicate within acidic and cathepsin D-positive vacuolar phagolysosomes.     

Identification of an intracellular M17 family leucine aminopeptidase that is required for virulence in Staphylococcus aureus

Staphylococcus aureus is a highly virulent bacterial pathogen capable of causing a variety of ailments throughout the human body. It is a major public health concern due to the continued emergence of highly pathogenic methicillin resistant strains (MRSA) both within hospitals and in the community. Virulence in S. aureus is mediated by an array of secreted and cell wall associated virulence factors, including toxins, hemolysins and proteases. In this work we identify a leucine aminopeptidase (LAP, pepZ) that strongly impacts the pathogenic abilities of S. aureus. Disruption of the pepZ gene in either Newman or USA300 resulted in a dramatic attenuation of virulence in both localized and systemic models of infection. LAP is required for survival inside human macrophages and gene expression analysis shows that pepZ expression is highest in the intracellular environment. We examine the cellular location of LAP and demonstrate that it is localized to the bacterial cytosol. These results identify for the first time an intracellular leucine aminopeptidase that influences disease causation in a Gram-positive bacterium.