Staphylococcus aureus small colony variants impair host immunity by activating host cell glycolysis and inducing necroptosis

Staphylococcus aureus small colony variants (SCVs) are frequently associated with chronic infection, yet they lack expression of many virulence determinants associated with the pathogenicity of wild-type strains. We found that both wild-type S. aureus and a ΔhemB SCV prototype potently activate glycolysis in host cells. Glycolysis and the generation of mitochondrial reactive oxygen species were sufficient to induce necroptosis, a caspase-independent mechanism of host cell death that failed to eradicate S. aureus and instead promoted ΔhemB SCV pathogenicity. To support ongoing glycolytic activity, the ΔhemB SCV induced over a 100-fold increase in the expression of fumC, which encodes an enzyme that catalyses the degradatin of fumarate, an inhibitor of glycolysis. Consistent with fumC-dependent depletion of local fumarate, the ΔhemB SCV failed to elicit trained immunity and protection from a secondary infectious challenge in the skin. The reliance of the S. aureus SCV population on glycolysis accounts for much of its role in the pathogenesis of S. aureus skin infection

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance. Keywords: immunometabolism, Klebsiella pneumoniae, immunosuppressive, anti-inflammatory, itaconate, Type Six Secretion Syste

Characterisation of a family of novel glycosyltransferases from enteropathogenic Escherichia coli and Salmonella

© 2016 Dr. Tania Wong Fok LungEnteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen of children that utilises a type III secretion system (T3SS) to inject virulence effector proteins into enterocytes during infection. NleB1 is a novel glycosyltransferase effector from EPEC that catalyses the addition of a single GlcNAc moiety in an N-glycosidic linkage to arginine. NleB1 modifies arginine-117 (Arg117) of the Fas associated death domain (DD) protein, FADD, which prevents assembly of the canonical death inducing signalling complex (DISC) and inhibits FasL-induced cell death. NleB1 also modifies the equivalent arginine residues in the DD proteins, tumour necrosis factor receptor type 1 (TNFR1)-associated death domain (TRADD) and receptor-interacting protein kinase 1 (RIPK1). Apart from the DxD catalytic motif of NleB1, little is known about other functional sites in the protein and the regions required for substrate binding and specificity. Here a library of 22 random transposon-based, in-frame, linker insertion mutants of NleB1 were tested for their ability to block caspase-8 activation in response to FasL during EPEC infection. Immunoblot analysis of caspase-8 cleavage products showed that 14 mutant derivatives of NleB1 no longer inhibited caspase-8 activation, including the catalytic DxD mutant. Regions of interest around the linker insertion sites were examined further with multiple or single amino acid substitutions. Coimmunoprecipitation studies of 34 site-directed mutants showed that the NleB1 derivatives with the E253A, Y219A, and PILN(63– 66)AAAA (in which the PILN motif from residues 63 to 66 was changed to AAAA) mutations bound to but did not GlcNAcylate FADD. A further mutant derivative, the PDG(236 –238)AAA mutant, did not bind to or GlcNAcylate FADD. Further testing of these mutants with TRADD and RIPK1, showed that NleB1 bearing the mutations E253A and Y219A could still bind to FADD and RIPK1 but not to TRADD. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleBE253A and NleBY219A showed that these 2 strains were attenuated, indicating the importance of the residues E253 and Y219 in NleB1 virulence in vivo. In summary, we identified new amino acid residues critical for NleB1 activity and confirmed that FADD GlcNAcylation was critical for NleB1 function. Close homologues of NleB1 are found in Salmonella enterica serovar Typhimurium and these are termed SseK1, SseK2 and SseK3. We hypothesized that the SseK effectors would also bind to DD proteins and inhibit apoptotic or inflammatory signalling. The SseK effectors did not appear to play a strong role in the inhibition of death receptor signaling given that we could not detect binding of the SseK effectors to the death domain proteins FADD, TRADD and RIPK1, which are targets of NleB1. A further survey of DD proteins revealed that SseK3 bound to TNFR1. However S. Typhimurium did not appear to inhibit TNF-induced IL-8 production and the biological significance of this interaction is still unknown. We conclude that the SseKs have an alternative function during S. Typhimurium infection to NleB1 in EPEC

Metabolic Stress Drives Keratinocyte Defenses against Staphylococcus aureus Infection

Human skin is commonly colonized and infected by Staphylococcus aureus. Exactly how these organisms are sensed by keratinocytes has not been clearly delineated. Using a combination of metabolic and transcriptomic methodologies, we found that S. aureus infection is sensed as a metabolic stress by the hypoxic keratinocytes. This induces HIF1α signaling, which promotes IL-1β production and stimulates aerobic glycolysis to meet the metabolic requirements of infection. We demonstrate that staphylococci capable of glycolysis, including WT and agr mutants, readily induce HIF1α responses. In contrast, Δpyk glycolytic mutants fail to compete with keratinocytes for their metabolic needs. Suppression of glycolysis using 2-DG blocked keratinocyte production of IL-1β in vitro and significantly exacerbated the S. aureus cutaneous infection in a murine model. Our data suggest that S. aureus impose a metabolic stress on keratinocytes that initiates signaling necessary to promote both glycolysis and the proinflammatory response to infection

The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.

Enteropathogenic Escherichia coli (EPEC) is a gastrointestinal pathogen that utilizes a type III secretion system (T3SS) to inject an array of virulence effector proteins into host enterocytes to subvert numerous cellular processes for successful colonization and dissemination. The T3SS effector NleD is a 26-kDa zinc metalloprotease that is translocated into host enterocytes, where it directly cleaves and inactivates the mitogen-activated protein kinase signaling proteins JNK and p38. Here a library of 91 random transposon-based, in-frame, linker insertion mutants of NleD were tested for their ability to cleave JNK and p38 during transient transfection of cultured epithelial cells. Immunoblot analysis of p38 and JNK cleavage showed that 7 mutant derivatives of NleD no longer cleaved p38 but maintained the ability to cleave JNK. Site-directed mutation of specific regions surrounding the insertion sites within NleD revealed that a single amino acid, R203, was essential for cleavage of p38 but not JNK in a direct in vitro cleavage assay, in transiently transfected cells, or in EPEC-infected cells. Mass spectrometry analysis narrowed the cleavage region to within residues 187 and 213 of p38. Mutation of residue R203 within NleD to a glutamate residue abolished the cleavage of p38 and impaired the ability of NleD to inhibit AP-1-dependent gene transcription of a luciferase reporter. Furthermore, the R203 mutation abrogated the ability of NleD to dampen interleukin-6 production in EPEC-infected cells. Overall, this work provides greater insight into substrate recognition and specificity by the type III effector NleD

SseK3 is a salmonella effector that binds trim32 and modulates the host's nf-kappa b signalling activity

Salmonella Typhimurium employs an array of type III secretion system effectors that facilitate intracellular survival and replication during infection. The Salmonella effector SseK3 was originally identified due to amino acid sequence similarity with NleB; an effector secreted by EPEC/EHEC that possesses N-acetylglucoasmine (GlcNAc) transferase activity and modifies death domain containing proteins to block extrinsic apoptosis. In this study, immunoprecipitation of SseK3 defined a novel molecular interaction between SseK3 and the host protein, TRIM32, an E3 ubiquitin ligase. The conserved DxD motif within SseK3, which is essential for the GlcNAc transferase activity of NleB, was required for TRIM32 binding and for the capacity of SseK3 to suppress TNF-stimulated activation of NF-kappa B pathway. However, we did not detect GlcNAc modification of TRIM32 by SseK3, nor did the SseK3-TRIM32 interaction impact on TRIM32 ubiquitination that is associated with its activation. In addition, lack of sseK3 in Salmonella had no effect on production of the NF-kappa B dependent cytokine, IL-8, in HeLa cells even though TRIM32 knockdown suppressed TNF-induced NF-kappa B activity. Ectopically expressed SseK3 partially co-localises with TRIM32 at the trans-Golgi network, but SseK3 is not recruited to Salmonella induced vacuoles or Salmonella induced filaments during Salmonella infection. Our study has identified a novel effector-host protein interaction and suggests that SseK3 may influence NF-kappa B activity. However, the lack of GlcNAc modification of TRIM32 suggests that SseK3 has further, as yet unidentified, host targets

Ectopic expression of SseK3 does not alter the ubiquitination of TRIM32.

<p>Sub-confluent A431 cells were transiently co-transfected with plasmids encoding Myc-TRIM32 and GFP-SseK3 or GFP-SseK3AAA. Untransfected cells were included as a negative control. 16–18 hrs post transfections, cells were then washed and harvested in TK lysis buffer. Equal amounts of pre-cleared cell lysates were used for immunoprecipitation using mouse monoclonal anti-Myc antibody coupling with Protein G agarose beads. Immunoprecipitated proteins <b>(A)</b> and whole cell lysates <b>(B)</b> were boiled for 5 min in SDS sample loading buffer and resolved by SDS-PAGE/ western blots. Membranes were incubated with anti-ubiquitin or anti-Myc antibodies, whereas anti-tubulin antibody was included as a loading control. After the incubation with IRDye conjugated fluorescence secondary antibodies, fluorescence intensities were detected and scanned by using Li-COR Odyssey infrared imaging system. Represented images from three independent experiments were shown. The asterix (*) indicates 80 kDa of mono-ubiquitinated TRIM32.</p

GFP-SseK3 preferentially interacts with non-ubiquitinated TRIM32.

<p>Sub-confluent A431 cells were transiently transfected with plasmids encoding GFP-SseK1, GFP-SseK2, GFP-SseK3 or Myc-TRIM32 alone, or both GFP-SseK and Myc-TRIM32. 16–18 h post transfection, cells were washed with chilled PBS and lysed on ice using TK lysis buffer. Equal amount of cell lysates were used for the immunoprecipitation using GFP-nanotrap beads. Immunoprecipitated complexes <b>(A)</b> and cell lysates <b>(B)</b> were resuspended in SDS sample loading buffer and boiled for 5 min before resolved by SDS-PAGE, and transferred onto Immobilon-FL membrane. After blocking in Odyssey blocking buffer, membranes were incubated with primary antibodies against GFP and Myc, followed by IRDye conjugated secondary antibodies. Fluorescence was detected by Li-COR Odyssey infrared imaging system. Untransfected cells were used as a negative control. Representative images from three independent experiments are shown.</p

SseK3AAA mutant has reduced affinity for Myc-TRIM32.

<p>Sub-confluent A431 cells were transiently co-transfected with plasmids encoding Myc-TRIM32 and GFP-SseK3 or GFP-SseK3AAA. Untransfected cells were included as a negative control. 16–18 h post transfection, cells were then washed and harvested in TK lysis buffer. Equal amounts of pre-cleared cell lysates were used for immunoprecipitation using mouse monoclonal anti-Myc antibody coupling with Protein G agarose beads. Immunoprecipitated proteins <b>(A)</b> and whole cell lysates <b>(B)</b> were boiled for 5 min in SDS sample loading buffer and resolved by SDS-PAGE/ western blots. Membranes were incubated with anti-GFP or anti-Myc antibodies, whereas anti-tubulin antibody was included as a loading control. After incubation with IRDye conjugated fluorescence secondary antibodies, fluorescence intensities were detected and scanned by using Li-COR Odyssey infrared imaging system. Representative images from three independent experiments are shown.</p