Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of β2 integrin‐mediated signalling pathway

Establishment of infection by facultative intracellular pathogen Mycobacterium tuberculosis (Mtb) requires adherence to and internalisation by macrophages. However, the effector molecules exploited by Mtb for entry into macrophages remain to be fully understood. The mammalian cell entry (Mce) proteins play an essential role in facilitating the internalisation of mycobacteria into mammalian cells. Here, we characterized Mtb Mce3C as a new mycobacterial surface protein that could promote mycobacterial adhesion to and invasion of macrophages in an RGD motif‐dependent manner. We then further demonstrated that β2 integrin was required for Mce3C‐mediated cell entry. In addition, we found that binding of Mce3C recruited β2 integrin‐dependent signalling adaptors and induced local actin rearrangement at the site of mycobacterial invasion. By using specific antibodies and pharmacological inhibitors, we further demonstrated the involvement of Src‐family tyrosine kinases, spleen tyrosine kinase, Vav, Rho, and Rho‐associated kinase in Mce3C‐mediated mycobacterial invasion. Our results reveal a novel mechanism by which Mtb Mce3C exploits integrin‐mediated signalling cascade for Mce, providing potential targets for the development of therapies against Mtb infection.     

The C‐type lectin receptor MGL senses N‐acetylgalactosamine on the unique Staphylococcus aureus ST395 wall teichoic acid

Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern‐recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved β‐1,4‐linked N‐acetylglucosamine (GlcNAc) modification on S. aureus wall teichoic acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase‐negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly‐glycerolphosphate with α‐O‐N‐acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage galactose‐type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN‐ and GalNAc‐dependent manner but did not interact with different tagN‐positive CoNS species. However, heterologous expression of Staphylococcus lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc‐transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte‐derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance.     

Canonical and noncanonical inflammasomes in intestinal epithelial cells

Inflammasomes are cytosolic, multimeric protein complexes capable of activating pro‐inflammatory cytokines such as IL‐1β and IL‐18, which play a key role in host defence. Inflammasome components are highly expressed in the intestinal epithelium. In recent years, studies have begun to demonstrate that epithelial‐intrinsic inflammasomes play a critical role in regulating epithelial homeostasis, both by defending the epithelium from pathogenic insult and through the regulation of the mucosal environment. However, the majority of research regarding inflammasome activation has focused on professional immune cells, such as macrophages. Here, we present an overview of the current understanding of inflammasome function in epithelial cells and at mucosal surfaces and, in particular, in the intestine.     

Effect of pharynx epithelial cells surface desialylation on receptor‐mediated adherence of Staphylococcus aureus

Aims: To characterize the interaction between cell surface carbohydrates and Staphylococcus aureus. Methods and Results: In the present study, in vitro adherence of S. aureus to Detroit 562 cells, amount of cell surface desialylation and effect of subterminal monosaccharides on desialylated glycoproteins on adherence was studied with colony counting, HPLC, fluorescence microscopy and fluorometric techniques. According to our findings, S. aureus adherence to pharynx cells was enhanced (40%) after neuraminidase treatment, and neuraminidase also cleave great amount of Detroit 562 cells surface sialic acid (39–60%). Adherence assay with various monosaccharides‐pretreated bacteria, and lectin competitive inhibition, showed that the residual subterminal galactose, fucose and N‐acetyl‐d ‐glucosamine remaining on desialylated Detroit 562 cell surface glycoproteins responsible for this binding. Conclusion: The results are the first to show that galactose, fucose and N‐acetyl‐d ‐glucosamine remaining on desialylated pharynx cell surface glycoproteins serve as the adhesine receptors for S. aureus. Significance and Impact of the Study: This study may explain the predisposition of severe S. aureus pneumonia complication in respiratory viral infections.     

A novel in vitro model for haematogenous spreading of S. aureus device biofilms demonstrating clumping dispersal as an advantageous dissemination mechanism

Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life‐threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics in the process. Using an in vitro catheter infection model, we studied S. aureus biofilm growth, late‐stage dispersal, and reattachment to downstream endothelial cell layers. The ability of the released biofilm material to resist host response and disseminate in vivo was furthermore studied in whole blood and phagocyte survival assays and in a short‐term murine infection model. We found that S. aureus biofilms formed in flow of human plasma release biofilm thromboemboli with embedded bacteria and bacteria‐secreted polysaccharides. The emboli disseminate as antibiotic and immune resistant vehicles that hold the ability to adhere to and initiate colonisation of endothelial cell layers under flow. In vivo experiments showed that the released biofilm material reached the heart similarly as ordinary broth‐grown bacteria but also that clumps to some extend were trapped in the lungs. The clumping dispersal of S. aureus from in vivo‐like vascular biofilms and their specific properties demonstrated here help explain the pathophysiology associated with S. aureus bloodstream infections.     

Contaminations of laboratory surfaces with Staphylococcus aureus are affected by the carrier status of laboratory staff

Aim: As a biosafety laboratory, we take samples from surfaces in microbiological laboratories to survey the handling of micro‐organisms. Whereas contaminations with other micro‐organisms were rare, Staphylococcus aureus was found in the working environment of many laboratories. As 20–60% of the healthy population are carriers of S. aureus we wanted to asses the effect of carriers on our sampling results. Methods and Results: Nasal swabs of staff members in nonmicrobiological laboratories and offices as well as surface samples from their personal work environment were taken and analysed for S. aureus DNA. In addition S. aureus strains were isolated using S. aureus‐specific agar plates and analysed by randomly amplified polymorphic DNA (RAPD)–PCR and multilocus sequence typing (MLST). Our data show that contaminations with S. aureus in nonmicrobiological environments are common with 29% of the surface samples containing S. aureus DNA. In the working environment of carriers, the number of contaminations was significantly increased compared to the environment of noncarriers. Conclusion: The carrier status of staff members significantly affects the number of contaminations on laboratory surfaces. Therefore, even in the absence of intentional handling of S. aureus, contaminations can be detected on a substantial amount of surfaces. Significance and Impact of the Study: Sampling procedures need to be adapted based on these results with respect to the locations where samples are taken and the threshold for significant contaminations. Because of its wide distribution, S. aureus can serve as a marker for hygienic standards in laboratories.     

Citrobacter rodentium induces rapid and unique metabolic and inflammatory responses in mice suffering from severe disease

The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell‐based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP‐heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections.     

Increased tolerance of Staphylococcus aureus to vancomycin in viscous media

The increased viscosity observed in biofilms, adherent communities of bacterial cells embedded in a polymeric matrix, was hypothesized to induce increased tolerance of bacteria to antibiotics. To test this concept, planktonic Staphylococcus aureus cells were grown and exposed to vancomycin in media brought to specific viscosities in order to mimic the biofilm extracellular polymeric matrix. A viscous environment was observed to decrease the vancomycin susceptibility of planktonic S. aureus to levels seen for biofilms. Both planktonic S. aureus at a viscosity of 100 mPa s and staphylococcal biofilms were able to survive at >500 times the levels of the antibiotic effective against planktonic populations in standard medium. Time-dependent and dose-dependent viability curves revealed that more than one mechanism was involved in high S. aureus tolerance to vancomycin in viscous media. Increased viscosity affects antibiotic susceptibility by reducing diffusion and the mass transfer rate; this mechanism alone, however, cannot explain the increased tolerance demonstrated by S. aureus in viscous media, suggesting that viscosity may also alter the phenotype of the planktonic bacteria to one more resistant to antimicrobials, as seen in biofilms. However, these latter changes are not yet understood and will require further study.     

Interferon‐gamma production in Lyme arthritis synovial tissue promotes differentiation of fibroblast‐like synoviocytes into immune effector cells

Lyme arthritis (LA), a late disease manifestation of Borrelia burgdorferi infection, usually resolves with antibiotic therapy. However, some patients develop proliferative synovitis lasting months to several years after spirochetal killing, called postinfectious LA. In this study, we phenotyped haematopoietic and stromal cell populations in the synovial lesion ex vivo and used these findings to generate an in vitro model of LA using patient‐derived fibroblast‐like synoviocytes (FLS). Ex vivo analysis of synovial tissue revealed high abundance of IFNγ‐producing T cells and NK cells. Similar to marked IFNγ responses in tissue, postinfectious LA synovial fluid also had high levels of IFNγ. HLA‐DR‐positive FLS were present throughout the synovial lesion, particularly in areas of inflammation. FLS stimulated in vitro with B. burgdorferi, which were similar to conditions during infection, expressed 68 genes associated primarily with innate immune activation and neutrophil recruitment. In contrast, FLS stimulated with IFNγ, which were similar to conditions in the postinfectious phase, expressed >2,000 genes associated with pathogen sensing, inflammation, and MHC Class II antigen presentation, similar to the expression profile in postinfectious synovial tissue. Furthermore, costimulation of FLS with B. burgdorferi and IFNγ induced greater expression of IL‐6 and other innate immune response proteins and genes than with IFNγ stimulation alone. These results suggest that B. burgdorferi infection, in combination with IFNγ, initiates the differentiation of FLS into a highly inflammatory phenotype. We hypothesise that overexpression of IFNγ by lymphocytes within synovia perpetuates these responses in the postinfectious period, causing proliferative synovitis and stalling appropriate repair of damaged tissue.     

The effect of acquired resistance to mercury on the phage sensitivity pattern of Staphylococcus aureus and its adherence to buccal epithelial cells

Abstract The study on the effect of acquired resistance to mercury on the phage sensitivity pattern of Staphylococcus aureus and its adherence to buccal epithelial cells showed increased lag period in growth and inverse relationship between the mercury resistance and cell wall teichoic acid, protein A and binding of cells to normal humal buccal cells. The phage pattern was found to change with the development of mercury resistance.     

Leucocyte recruitment and molecular fortification of keratinocytes triggered by streptococcal M1 protein

Streptococcus pyogenes of the M1 serotype is commonly associated with invasive streptococcal infections and development of streptococcal toxic shock syndrome. The M1 protein is a powerful inducer of inflammatory responses for several human cell types, but the reason why M1 protein‐related strains is over‐represented in invasive streptococcal diseases is still not understood. This study was undertaken to investigate if soluble M1 protein can aggravate the severity of streptococcal skin infections in respect to inflammation, leucocyte recruitment, and tissue remodelling as seen in patients with cellulitis and necrotizing fasciitis. We found that HaCaT cells are able to recruit activated leucocytes when encountering M1 protein. Neither the bacterial protein nor activated leucocytes caused cell damage on HaCaT cells, instead HaCaT cells responded to the bacterial virulence factor by releasing several proteins protective against bacterial infection and leucocyte responses. However, although not cytotoxic, M1 protein completely abolished wound healing abilities of HaCaT cells. Taken together, our results demonstrate that M1 protein is a critical virulence factor that can augment streptococcal skin infection suggesting that the protein is an interesting target for drug development.     

Plasmid curing in Staphylococcus aureus by antibiotics affecting the bacterial cell wall

Abstract Strains of Staphylococcus aureus were converted into L-forms with β-lactam antibiotics, vancomycin and lysostaphin. Reverted bacteria obtained after several transfers in protoplast forms (unstable L-forms) as well as stable L-forms lost their plasmids. Curing was obtained whatever the plasmid size (from 3.4 to 28.2 kb) but complete curing required cell division. Elimination of plasmids in wall-less organisms could be the result of an inhibition of new rounds of plasmid replication following the loss of DNA-envelope interactions.     

Potential action of copper surfaces on meticillin-resistant Staphylococcus aureus

Aims: Studies to date have shown rapid killing of bacterial cells when exposed to copper surfaces. The mechanistic action of copper on bacterial cells is so far unknown. Methods and Results: To investigate potential mechanisms involved, meticillin‐resistant Staphylococcus aureus (MRSA) cells (10⁷ CFU) were inoculated onto coupons of copper or stainless steel and stained with either the viability fluorophore 5‐cyano‐2,3‐ditolyl tetrazolium (CTC), to detect respiration, or BacLight? (SYTO9/propidium iodide), to determine cell wall integrity. Coupons were then observed in‐situ using epifluorescence microscopy. In addition, DNA from cells inoculated onto either copper or stainless steel surfaces was isolated and analysed by agarose gel electrophoresis. An effect on cellular respiration with CTC reduction was evident but no effect on cell membrane integrity (BacLight?) was observed. Results from the DNA isolation indicated a copper‐induced detrimental effect on MRSA genomic material as no bands were observed after exposure to copper surface. Conclusions: The results indicate that exposure to copper surfaces rapidly kills MRSA by compromising cellular respiration and damaging DNA, with little effect on cell membrane integrity. Significance and Impact of the study: This research provides a mechanistic explanation in support of previous suggestions that although copper surfaces do not affect membrane integrity of cells, there is still a rapid antimicrobial effect.     

TLR2-mediated survival of Staphylococcus aureus in macrophages: a novel bacterial strategy against host innate immunity

TLR2 plays a role as a pattern-recognition receptor in the innate immune response involving secreted proteins against microbial pathogens. To examine its possible involvement in the cellular response, we determined the levels of the engulfment and subsequent killing of bacteria by macrophages prepared from TLR2-deficient and wild-type mice. The level of the engulfment of Staphylococcus aureus or Escherichia coli was almost the same between TLR2-lacking and wild-type macrophages. However, the colony-forming ability of engulfed S. aureus, but not of E. coli, decreased to a greater extent in TLR2-lacking macrophages than in the wild-type control. The incubation with S. aureus caused activation of JNK in wild-type macrophages but not in TLR2-lacking macrophages, and the pretreatment of wild-type macrophages with a JNK inhibitor increased the rate of killing of engulfed S. aureus, but again not of E. coli. In addition, the number of colonies formed by engulfed S. aureus increased in the JNK-dependent manner when TLR2-lacking macrophages were pretreated with LPS. Furthermore, JNK seemed to inhibit the generation of superoxide, not of NO, in macrophages. These results collectively suggested that the level of superoxide is reduced in macrophages that have engulfed S. aureus through the actions of TLR2-activated JNK, resulting in the prolonged survival of the bacterium in phagosomes. The same regulation did not influence the survival of E. coli, because this bacterium was more resistant to superoxide than S. aureus. We propose a novel bacterial strategy for survival in macrophages involving the hijacking of an innate immune receptor.