Identification of Shigella flexneri IcsA residues affecting interaction with N-WASP, and evidence for IcsA-IcsA co-operative interaction

The Shigella flexneri IcsA (VirG) protein is a polarly distributed outer membrane protein that is a fundamental virulence factor which interacts with neural Wiskott-Aldrich syndrome protein (N-WASP). The activated N-WASP then activates the Arp2/3 complex which initiates de novo actin nucleation and polymerisation to form F-actin comet tails and allows bacterial cell-to-cell spreading. In a previous study, IcsA was found to have three N-WASP interacting regions (IRs): IR I (aa 185–312), IR II (aa 330–382) and IR III (aa 508–730). The aim of this study was to more clearly define N-WASP interacting regions II and III by site-directed mutagenesis of specific amino acids. Mutant IcsA proteins were expressed in both smooth lipopolysaccharide (S-LPS) and rough LPS (R-LPS) S. flexneri strains and characterised for IcsA production level, N-WASP recruitment and F-actin comet tail formation. We have successfully identified new amino acids involved in N-WASP recruitment within different N-WASP interacting regions, and report for the first time using co-expression of mutant IcsA proteins, that N-WASP activation involves interactions with different regions on different IcsA molecules as shown by Arp3 recruitment. In addition, our findings suggest that autochaperone (AC) mutant protein production was not rescued by another AC region provided in trans, differing to that reported for two other autotransporters, PrtS and BrkA autotransporters

The role of bacterial protein tyrosine phosphatases in the regulation of the biosynthesis of secreted polysaccharides

SIGNIFICANCE: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria.RECENT ADVANCES: Deletion and over-expression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases has suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. CRITICAL ISSUES: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. FUTURE DIRECTIONS: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel anti-microbials. This is particularly the case for the polymerase and histidinol phosphatase family, which are predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism and critical importance of these phosphatases in a wide range of bacteria.Alistair James Standish and Renato Moron

Dynamin-related protein Drp1 and mitochondria are important for Shigella flexneri infection

Abstract unavailable.Mabel Lum, Renato Moron

Myosin IIA is essential for Shigella flexneri cell-to-cell spread

First published online: 8 January 2015A key feature of Shigella pathogenesis is the ability to spread from cell-to-cell post-invasion. This is dependent on the bacteria's ability to initiate de novo F-actin tail polymerisation, followed by protrusion formation, uptake of bacteria-containing protrusion and finally, lysis of the double membrane vacuole in the adjacent cell. In epithelial cells, cytoskeletal tension is maintained by the actin-myosin II networks. In this study, the role of myosin II and its specific kinase, myosin light chain kinase (MLCK), during Shigella intercellular spreading was investigated in HeLa cells. Inhibition of MLCK and myosin II, as well as myosin IIA knockdown, significantly reduced Shigella plaque and infectious focus formation. Protrusion formation and intracellular bacterial growth was not affected. Low levels of myosin II were localised to the Shigella F-actin tail. HeLa cells were also infected with Shigella strains defective in cell-to-cell spreading. Unexpectedly loss of myosin IIA labelling was observed in HeLa cells infected with these mutant strains. This phenomenon was not observed with WT Shigella or with the less abundant myosin IIB isoform, suggesting a critical role for myosin IIA.Mabel Lum, Renato Moron

Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy; identification of the residues affecting O antigen modal chain length control, and Wzz dependent polymerisation activity

Published Online: 01/04/2015The O antigen (Oag) component of lipopolysaccharide (LPS) is a major S. flexneri virulence determinant. Oag is polymerised by WzySf, and its modal chain length is determined by WzzSf and WzzpHS2. Site-directed mutagenesis was performed on wzySf in pWaldo-wzySf-TEV-GFP to alter Arg residues in WzySf's two large periplasmic loops (PL) (PL3 and PL5). Analysis of the LPS profiles conferred by mutated WzySf proteins in the wzySf deficient (Δwzy) strain identified residues that affect WzySf activity. The importance of the guanidium group of the Arg residues was investigated by altering the Arg residues to Lys and Glu, which generated WzySf mutants conferring altered LPS Oag modal chain lengths. The dependence of these WzySf mutants on WzzSf was investigated by expressing them in a wzySf and wzzSf deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles identified a role for the Arg residues in the association of WzySf and WzzSf during Oag polymerisation. Colicin E2 and bacteriophage Sf6c susceptibility supported this conclusion. Comparison of the expression levels of different mutant WzySf-GFP proteins with the wild type (WT) WzySf-GFP showed that certain Arg residues affected production levels of WzySf in a WzzSf dependent manner. To our knowledge, this is the first report of S. flexneri WzySf mutants having an effect on LPS Oag modal chain length, and identified functionally significant Arg residues in WzySf.Pratiti Nath and Renato Moron

Lungfishes, Like Tetrapods, Possess a Vomeronasal System

The vomeronasal system (VNS) is an accessory olfactory system that in tetrapod vertebrates is composed of specific receptor neurons in the nasal organ and a set of centers in the forebrain that receive and relay the information consecutively towards the hypothalamus. Thus, only in tetrapods the VNS comprises a discrete vomeronasal (Jacobson's) organ, which contains receptor cells that are morphologically distinct from those of the olfactory epithelium and use different transduction mechanisms. The axons of the vomeronasal receptors in tetrapods project to the accessory olfactory bulb (AOB) in the rostral telencephalon. Secondary vomeronasal connections exist through the medial amygdala to the hypothalamus. Currently, the lungfishes are considered the closest living relatives of tetrapods. Here we show that the African lungfish, Protopterus dolloi, has epithelial crypts at the base of the lamellae of the olfactory epithelium that express markers of the vomeronasal receptors in tetrapods. The projections of these crypts allow us to identify an AOB on the lateral margin of the main olfactory bulb. The projections of this AOB reach a region that is topologically, hodologically, and immunohistochemically identical to the medial amygdala and could represent its homolog. Neurons of this putative medial amygdala were demonstrated to project to the lateral hypothalamus, as they do in tetrapods. All these features that lungfishes share with tetrapods indicate that lungfishes have the complete set of brain centers and connections involved in processing vomeronasal information and that these features were already present in the last common ancestor of lungfishes and tetrapods

Relationship between O-antigen chain length and resistance to colicin E2 in Shigella flexneri

The Shigella flexneri polysaccharide co-polymerase class 1a (PCP1a) protein, WzzBSF, regulates LPS O-antigen (Oag) chain length to confer short (S)-type Oag chains of ~10–17 Oag repeat units (RUs). The S-type Oag chains affect Shigella flexneri virulence as they influence IcsA-mediated actin-based motility. However, they do not confer resistance to complement; this is conferred by the very-long (VL)-type Oag chains determined by WzzBpHS2. Colicins are bacterial proteins produced by some Escherichia coli strains to kill related strains. While the presence of Oag chains has been shown to shield outer-membrane proteins from colicins, the impact of Oag chain length against colicins is unknown. In this study, initial testing indicated that a Shigella flexneri Y wzz : : kanr mutant was more sensitive to colicin E2 compared with the WT strain. Plasmids encoding Wzz mutant and WT PCP1a proteins conferring different Oag modal chain lengths were then expressed in the mutant background, and tested against purified colicin E2. Analysis of swab and spot sensitivity assays showed that strains expressing either S-type or long (L)-type Oag chains (16–28 Oag RUs) conferred greater resistance to colicin E2 compared with strains having very-short-type (2–8 Oag RUs), intermediate-short-type (8–14 Oag RUs) or VL-type (>80 Oag RUs) Oag chains. These results suggest a novel role for LPS Oag chain length control that may have evolved due to selection pressure from colicins in the environment.Elizabeth Ngoc Hoa Tran, Magdalene Papadopoulos and Renato Moron

Bacteriophage Sf6 host range mutant that infects Shigella flexneri serotype 2a(2) strains

Shigella flexneri serotype 2a2 (II:9;10) is the most prevalent strain in causing bacillary dysentery in developing countries. Chemical modifications such as glucosylation, O-acetylation, and phosphoethanolamine modifications of lipopolysaccharide (LPS) O antigen (Oag) contribute to the emergence of various serotypes. Sf6 is a Shigella-specific bacteriophage that infects only a limited range of S. flexneri serotypes [X, Y]. LPS Oag is the primary receptor for bacteriophage Sf6 where it uses its tailspike protein (TSP) in binding and hydrolysing LPS Oags. Sf6TSP has recently been shown to be capable of hydrolysing the LPS Oag of Type II strains, albeit modestly. Phage therapy has regained attention in recent years as an alternative therapeutic approach. Therefore, this study aimed to expand the host range of Sf6 to the prevalent S. flexneri serotype 2a2 strain. We discovered a new lytic Sf6 host range mutant that is capable of infecting S. flexneri serotype 2a2 and identified residues in Sf6TSP that may potentially be involved in binding and hydrolysing serotype 2a2 LPS Oag. This work increased the limited Shigella-specific bacteriophage collection and may be useful in the future for phage therapy and/or biocontrolling of S. flexneri in contaminated food and water.Min Yan Teh, Elizabeth Ngoc Hoa Tran and Renato Moron

Progress in understanding the assembly process of bacterial O-antigen

The discovery that the surfaces of Gram-negative bacteria often carry unique polysaccharide signatures pre-dates most seminal discoveries of molecular biology and biochemistry of the 20th century. The O-antigen component of the lipopolysaccharide has been one of the most intensely studied bacterial polysaccharide surface structures for over 80 years. Yet, many questions about the mechanism of biosynthesis of the O-antigen and its transport to the cell surface remain unanswered. In this review we provide an overview of how the molecular basis of the O-antigen assembly and trafficking were unraveled in a historical context. We pay particular attention to the emergence of novel technological approaches and how they fueled the elucidation of the O-antigen maturation process. Moreover, we provide a brief perspective on the biosynthesis of enterobacterial common antigen and underline the similarities and differences between the pathways used to assemble these two surface polysaccharides. Finally, we highlight key discoveries that led to the understanding of the mechanistic basis of bacteriophage-induced O-antigen modifications. We place special emphasis on the regulation of the length of O-antigen polymers and provide a detailed overview of the models explaining the O-antigen length determination. Finally, we highlight outstanding questions that need to be addressed both structurally and functionally to advance our understanding of the O-antigen assembly, trafficking and export within cellular and molecular contexts.Sergei Kalynych, Renato Morona & Miroslaw Cygle

Topology of Streptococcus pneumoniae CpsC, a Polysaccharide Copolymerase and Bacterial Protein Tyrosine Kinase Adaptor Protein

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. In Streptococcus pneumoniae this transmembrane adaptor is CpsC, with the C-terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest CpsC has two transmembrane domains, with the N and C-termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal HA-tagged Cps2C protein in D39. Incubation of both protoplasts and membranes with the CP-B resulted in complete degradation of HA-Cps2C in all cases, indicating that the C-terminus of Cps2C was likely extra-cytoplasmic, and hence the protein's topology was not as predicted. Similar results were seen with membranes from TIGR4, indicating Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C-terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C-terminus by flow cytometry indicated that it was extra-cytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this affect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C-terminus flipping across the cytoplasmic membrane where it interacts with CpsD in order to regulate tyrosine kinase activity.Jonathan J. Whittall, Renato Morona, Alistair J. Standis