Search

The bacterial Type VI secretion system (T6SS) is a dynamic macromolecular structure that promotes inter‐ and intra‐species competition through the delivery of toxic effector proteins into neighbouring cells. The T6SS contains 14 well‐characterised core proteins necessary for effector delivery (TssA‐M, PAAR). In this study, we have identified a novel accessory component required for optimal T6SS activity in the opportunistic pathogen Serratia marcescens, which we name TagV. Deletion of tagV, which encodes an outer membrane lipoprotein, caused a reduction in the T6SS‐dependent antibacterial activity of S. marcescens Db10. Mutants of S. marcescens lacking the core component TssJ, a distinct outer membrane lipoprotein previously considered essential for T6SS firing, retained a modest T6SS activity that could be abolished through deletion of tagV. TagV did not interact with the T6SS membrane complex proteins TssL or TssM, but is proposed to bind to peptidoglycan, indicating that the mechanism by which TagV promotes T6SS firing differs from that of TssJ. Homologues of tagV were identified in several other bacterial genera, suggesting that the accessory function of TagV is not restricted to S. marcescens. Together, our findings support the existence of a second, TssJ‐independent mechanism for T6SS firing that is dependent upon the activity of TagV proteins. [ABSTRACT FROM AUTHOR]

Energy‐dependent translational throttle A (EttA) from Escherichia coli is a paradigmatic ABC‐F protein that controls the first step in polypeptide elongation on the ribosome according to the cellular energy status. Biochemical and structural studies have established that ABC‐F proteins generally function as translation factors that modulate the conformation of the peptidyl transferase center upon binding to the ribosomal tRNA exit site. These factors, present in both prokaryotes and eukaryotes but not in archaea, use related molecular mechanisms to modulate protein synthesis for heterogenous purposes, ranging from antibiotic resistance and rescue of stalled ribosomes to modulation of the mammalian immune response. Here, we review the canonical studies characterizing the phylogeny, regulation, ribosome interactions, and mechanisms of action of the bacterial ABC‐F proteins, and discuss the implications of these studies for the molecular function of eukaryotic ABC‐F proteins, including the three human family members. [ABSTRACT FROM AUTHOR]

While NLRC4-dependent sensing of intracellular Gram-negative pathogens such as Salmonella enterica serovar typhimurium is a beneficial host response, NLRC4-dependent sensing of the Pseudomonas aeruginosa type 3 secretion system (T3SS) has been shown to be involved in pathogenicity. In mice, different pathogen-associated microbial patterns are sensed by the combination of the NLRC4-inflammasome with different neuronal apoptosis inhibitory proteins (NAIPs). NAIP2 is involved in sensing PscI, an inner-rod protein of the P. aeruginosa T3SS. Surprisingly, only a single human NAIP (hNAIP) has been found. Moreover, there is no description of hNAIP-NLRC4 inflammasome recognition of T3SS inner-rod proteins in humans. Here, we show that the P. aeruginosa T3SS inner-rod protein PscI and needle protein PscF are both sensed by the hNAIPNLRC4 inflammasome in human macrophages and PBMCs from healthy donors, allowing caspase-1 and IL-1β maturation and resulting in a robust inflammatory response. TLR4 and TLR2 are involved in redundantly sensing these two T3SS components. [ABSTRACT FROM AUTHOR]

Background: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H2O) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H2O. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displayed a very moderate role in vivo, it was found, as a recombinant soluble form, to be endowed with folding activities in vitro. [ABSTRACT FROM AUTHOR]

Constitutive overproduction of the pump MexXY-OprM is recognized as a major cause of resistance to aminoglycosides, fluoroquinolones, and zwitterionic cephalosporins in Pseudomonas aeruginosa. In this study, 57 clonally unrelated strains recovered from non-cystic fibrosis patients were analyzed to characterize the mutations resulting in upregulation of the mexXY operon. Forty-four (77.2%) of the strains, classified as agrZ mutants were found to harbor mutations inactivating the local repressor gene (mexZ) of the mexXY operon (n = 33; 57.9%) or introducing amino acid substitutions in its product, MexZ (n = 11; 19.3%). These sequence variations, which mapped in the dimerization domain, the DNA binding domain, or the rest of the MexZ structure, mostly affected amino acid positions conserved in TetR-like regulators. The 13 remaining MexXY-OprM strains (22.8%) contained intact mexZ genes encoding wild-type MexZ proteins. Eight (14.0%) of these isolates, classified as agrW1 mutants, overexpressed the gene PA5471, which codes for the MexZ antirepressor ArmZ [corrected], with 5 strains exhibiting growth defects at 37°C and 44°C, consistent with mutations impairing ribosome activity. Interestingly, one agrW1 mutant appeared to harbor a 7-bp deletion in the coding sequence of the leader peptide, PA5471.1, involved in ribosome-dependent, translational attenuation of PA5471 expression. Finally, DNA sequencing and complementation experiments revealed that 5 (8.8%) strains, classified as agrW2 mutants, harbored single amino acid variations in the sensor histidine kinase of ParRS, a two-component system known to positively control mexXY expression. Collectively, these results demonstrate that clinical strains of P. aeruginosa exploit different regulatory circuitries to mutationally overproduce the MexXY-OprM pump and become multidrug resistant, which accounts for the high prevalence of MexXY-OprM mutants in the clinical setting.

Multidrug resistance has become a serious concern in the treatment of bacterial infections. A prominent role is ascribed to the active efflux of xenobiotics out of the bacteria by a tripartite protein machinery. The mechanism of drug extrusion is rather well understood, thanks to the X-ray structures obtained for the Escherichia coli TolC/AcrA/AcrB model system and the related Pseudomonas aeruginosa OprM/MexA/MexB. However, many questions remain unresolved, in particular the stoichiometry of the efflux pump assembly. On the basis of blue native polyacrylamide gel electrophoresis (BN-PAGE) (Wittig et al., Nat. Protoc. 2006, 1, 418-428), we analyzed the binding stoichiometry of both palmitylated and non-palmitylated MexA with the cognate partner OprM trimer at different ratios and detergent conditions. We found that β-octyl glucopyranoside (β-OG) detergent was not suitable for this technique. Then we proved that MexA has to be palmitylated in order to stabilized the complex formation with OprM. Finally, we provided evidence for a two by two (2, 4, 6, or upper) binding of palmitylated MexA per trimer of OprM., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.