EsiB, a novel pathogenic Escherichia coli secretory immunoglobulin A-binding protein impairing neutrophil activation.

UNLABELLED: In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site. IMPORTANCE: Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.

Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis.

The CXC chemokine receptor 2 (CXCR2) on neutrophils, which recognizes chemokines produced at the site of infection, plays an important role in antimicrobial host defenses such as neutrophil activation and chemotaxis. Staphylococcus aureus is a successful human pathogen secreting a number of proteolytic enzymes, but their influence on the host immune system is not well understood. Here, we identify the cysteine protease Staphopain A as a chemokine receptor blocker. Neutrophils treated with Staphopain A are unresponsive to activation by all unique CXCR2 chemokines due to cleavage of the N-terminal domain, which can be neutralized by specific protease inhibitors. Moreover, Staphopain A inhibits neutrophil migration towards CXCR2 chemokines. By comparing a methicillin-resistant S. aureus (MRSA) strain with an isogenic Staphopain A mutant, we demonstrate that Staphopain A is the only secreted protease with activity towards CXCR2. Although the inability to cleave murine CXCR2 limits in-vivo studies, our data indicate that Staphopain A is an important immunomodulatory protein that blocks neutrophil recruitment by specific cleavage of the N-terminal domain of human CXCR2.

Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways.

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.

Staphylococcus aureus metalloprotease aureolysin cleaves complement C3 to mediate immune evasion.

Complement is one of the first host defense barriers against bacteria. Activated complement attracts neutrophils to the site of infection and opsonizes bacteria to facilitate phagocytosis. The human pathogen Staphylococcus aureus has successfully developed ways to evade the complement system, for example by secretion of specific complement inhibitors. However, the influence of S. aureus proteases on the host complement system is still poorly understood. In this study, we identify the metalloprotease aureolysin as a potent complement inhibitor. Aureolysin effectively inhibits phagocytosis and killing of bacteria by neutrophils. Furthermore, we show that aureolysin inhibits the deposition of C3b on bacterial surfaces and the release of the chemoattractant C5a. Cleavage analyses show that aureolysin cleaves the central complement protein C3. Strikingly, there was a clear difference between the cleavages of C3 in serum versus purified conditions. Aureolysin cleaves purified C3 specifically in the α-chain, close to the C3 convertase cleavage site, yielding active C3a and C3b. However, in serum we observe that the aureolysin-generated C3b is further degraded by host factors. We pinpointed these factors to be factor H and factor I. Using an aureolysin mutant in S. aureus USA300, we show that aureolysin is essential and sufficient for C3 cleavage by bacterial supernatant. In short, aureolysin acts in synergy with host regulators to inactivate C3 thereby effectively dampening the host immune response.

Complement inhibition by gram-positive pathogens: molecular mechanisms and therapeutic implications.

The plasma proteins of the complement system are essential in the innate immune response against bacteria. Complement labels bacteria with opsonins to support phagocytosis and generates chemoattractants to attract phagocytes to the site of infection. In turn, bacterial human pathogens have evolved different strategies to specifically impair the complement response. Here, we review the large arsenal of complement inhibitors produced by the gram-positive pathogens Staphylococcus aureus and Group A Streptococcus. We discuss how these bacterial molecules provide us with new tools to treat both infectious and inflammatory disease conditions in humans.

Intracellular restriction of a productive noncytopathic coronavirus infection.

Virus infection in vitro can either result in a cytopathic effect (CPE) or proceed without visible changes in infected cells (noncytopathic infection). We are interested in understanding the mechanisms controlling the impact of coronavirus infection on host cells. To this end, we compared a productive, noncytopathic infection of murine hepatitis virus (MHV) strain A59 in the fibroblastlike cell line NIH 3T3 with cytopathic MHV infections. Infected NIH 3T3 cells could be cultured for up to 4 weeks without apparent CPE and yet produce virus at 10(7) to 10(8) PFU/ml. Using flow cytometry, we demonstrated that NIH 3T3 cells expressed as much MHV receptor CEACAM1 as other cell lines which die from MHV infection. In contrast, using quantitative reverse transcription-PCR and metabolic labeling of RNA, we found that the rate of viral RNA amplification in NIH 3T3 cells was lower than the rate in cells in which MHV induces a CPE. The rate of cellular RNA synthesis in contact-inhibited confluent NIH 3T3 cells was also lower than in cells permissive to cytopathic MHV infection. However, the induction of cellular RNA synthesis in growing NIH 3T3 cells did not result in an increase of either viral RNA amplification or CPE. Our results suggest that a specific, receptor CEACAM1-independent mechanism restricting coronaviral RNA synthesis and CPE is present in NIH 3T3 and, possibly, other cells with preserved contact inhibition.