IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface

Background: Autotransporters are attractive cell surface display vehicles as they lack complex adaptor proteins necessary for protein export. Recent reports have suggested that the native effector domain (α domain) and translocation domain (β domain) interact with each other to drive translocation of the effector domain to the outer membrane. In this report we compared the expression, surface localisation and folding of TEM-1 β-lactamase (Bla) and maltose binding protein (MalE or MBP) fused to either full length Shigella flexneri IcsA (IcsA) autotransporter or to the β domain alone (IcsAβ) to determine the contribution of the native IcsA α domain in presenting the fusion proteins on the surface of E. coli K-12 UT5600 (ΔompT). Results: Expression of IcsA-Bla was greater than IcsAβ-Bla. High levels of IcsA-MalE were detected but IcsAβ-MalE was not expressed. All fusion proteins other than IcsAβ-MalE were localised to the outer membrane and were detected on the surface of UT5600 via immunofluorescence microscopy. All bacteria expressing IcsA-MalE were labelled with both α-IcsA and α-MBP. UT5600 expressing IcsAβ-MalE was not labelled with α-MBP. A third of UT5600 expressing IcsA-Bla were detectable with α-Bla but only 5% of UT5600 (IcsAβ-Bla) were labelled with α-Bla. The correct folding of the Bla moiety when fused to IcsA and IcsAβ was also retained as UT5600 expressing either fusion protein exhibited a decreased zone of inhibition in the presence of ampicillin. UT5600 expressing IcsA-Bla was more resistant compared to UT5600 expressing IcsAβ-Bla. Conclusions: The export mechanism of autotransporters is not well understood but accumulating evidence suggest a critical role for the native effector or α domain in facilitating its own export via interactions with the translocation or β domain. This is the first report directly comparing expression of heterologous proteins fused to the full length IcsA autotransporter and fusion to the β domain alone. Protein expression and surface presentation of the fusion proteins were dramatically improved when fused to IcsA rather than IcsAβ. Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native α domain. This work also provides further evidence for a key interaction between the autotransporter α and β domains.Mabel Lum and Renato Moron

Residues located inside the Escherichia coli FepE protein oligomer are essential for lipopolysaccharide O-antigen modal chain length regulation

The Escherichia coli O157 : H7 FepE protein regulates lipopolysaccharide (LPS) O-antigen (Oag) chain length to confer a very long modal chain length of >80 Oag repeat units (RUs). The mechanism by which FepE regulates Oag modal chain length and the regions within it that are important for its function remain unclear. Studies on the structure of FepE show that the protein oligomerizes. However, the exact size of the oligomer is in dispute, further hampering our understanding of its mechanism. Guided by information previously obtained for regions known to be important for Oag modal chain length determination in the homologous Shigella flexneri WzzBSF protein, a set of FepE mutant constructs with single amino acid substitutions was created. Analysis of the resulting LPS conferred by these mutant His6-FepE proteins showed that amino acid substitutions of leucine 168 (L168) and aspartic acid 268 (D268) resulted in LPS with consistently shortened Oag chain lengths of <80 Oag RUs. Substitution of FepE’s transmembrane cysteine residues did not affect function. Chemical cross-linking experiments on mutant FepE proteins showed no consistent correlation between oligomer size and functional activity, and MS analysis of FepE oligomers indicated that the in vivo size of FepE is consistent with a maximum size of a hexamer. Our findings suggest that different FepE residues, mainly located within the internal cavity of the oligomer, contribute to Oag modal chain length determination but not the oligomeric state of the protein.Elizabeth Ngoc Hoa Tran and Renato Moron

Absence of O antigen suppresses Shigella flexneri IcsA autochaperone region mutations

The Shigella flexneri IcsA (VirG) protein is a polarly distributed autotransporter protein. IcsA functions as a virulence factor by interacting with the host actin regulatory protein N-WASP, which in turn activates the Arp2/3 complex, initiating actin polymerization. Formation of F-actin comet tails allows bacterial cell-to-cell spreading. Although various accessory proteins such as periplasmic chaperones and the β-barrel assembly machine (BAM) complex have been shown to be involved in the export of IcsA, the IcsA translocation mechanism remains to be fully elucidated. A putative autochaperone (AC) region (amino acids 634–735) located at the C-terminal end of the IcsA passenger domain, which forms part of the self-associating autotransporter (SAAT) domain, has been suggested to be required for IcsA biogenesis, as well as for N-WASP recruitment, based on mutagenesis studies. IcsAi proteins with linker insertion mutations within the AC region have a significant reduction in production and are defective in N-WASP recruitment when expressed in smooth LPS (S-LPS) S. flexneri. In this study, we have found that the LPS O antigen plays a role in IcsAi production based on the use of an rmlD (rfbD) mutant having rough LPS (R-LPS) and a novel assay in which O antigen is depleted using tunicamycin treatment and then regenerated. In addition, we have identified a new N-WASP binding/interaction site within the IcsA AC region.Min Yan Teh, Elizabeth Ngoc Hoa Tran and Renato Moron

Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production

Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both Gram-negative and –positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated Gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae¸ which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzydependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.Alistair J. Standish, Angela A. Salim, Hua Zhang, Robert J. Capon and Renato Moron

Dual inhibition of DNA polymerase PolC and protein tyrosine phosphatase CpsB uncovers a novel antibiotic target

Increasing antibiotic resistance is making the identification of novel antimicrobial targets critical. Recently, we discovered an inhibitor of protein tyrosine phosphatase CpsB, fascioquinol E (FQE), which unexpectedly inhibited the growth of Gram-positive pathogens. CpsB is a member of the polymerase and histidinol phosphate phosphatase (PHP) domain family. Another member of this family found in a variety of Gram-positive pathogens is DNA polymerase PolC. We purified the PHP domain from PolC (PolC(PHP)), and showed that this competes away FQE inhibition of CpsB phosphatase activity. Furthermore, we showed that this domain hydrolyses the 5'-p-nitrophenyl ester of thymidine-5'-monophosphate (pNP-TMP), which has been used as a measure of exonuclease activity. Finally, we showed that FQE not only inhibits the phosphatase activity of CpsB, but also ability of PolC(PHP) to catalyse the hydrolysis of pNP-TMP. This suggests that PolC may be the essential target of FQE, and that the PHP domain may represent an as yet untapped target for the development of novel antibiotics.Alistair J. Standish, Angela A. Salim, Robert J. Capon, Renato Moron

LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP(HA) was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP(HA) was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP(HA) was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP(HA) and IcsA showed that IcsP(HA) preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP(HA) in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP(HA) was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP(HA) detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.Elizabeth Ngoc Hoa Tran, Matthew Thomas Doyle, Renato Moron

The tolC locus of Escherichia coli K-12 : gene, protein and function / Renato Morona

Typescript (photocopy)xi, 115 leaves, [24] leaves of plates : ill. ; 30 cm.Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 198

Attachment of capsular polysaccharide to the cell wall of Streptococcus pneumoniae type 2 is required for invasive disease

The capacity of Streptococcus pneumoniae to produce capsular polysaccharide (CPS) is essential for virulence. The CPS biosynthesis proteins CpsB, CpsC, and CpsD function to regulate CPS production via tyrosine phosphorylation of CpsD. This mechanism of regulating CPS production is important for enabling S. pneumoniae to cause invasive disease. Here, we identify mutations affecting the attachment of CPS to the cell wall. These mutations were located in cpsC, such that CpsC functioned independently from CpsD tyrosine phosphorylation. These mutants produced WT levels of CPS, but were unable to cause bacteremia in mice after intranasal challenge. This finding suggests that cell-wall attachment of CPS is essential for invasive pneumococcal disease; production of WT levels of CPS alone is not sufficient. We also show that cpsB mutants, which lack the phosphotyrosine-protein phosphatase, produced less CPS than the WT strain, but attached substantially more CPS to their cell wall. Thus, the phosphorylated form of CpsD promotes attachment of CPS to the cell wall