Amanda E. Rossiter

From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis

Autotransporters are a superfamily of proteins that use the type V secretion pathway for their delivery to the surface of Gram-negative bacteria. At first glance, autotransporters look to contain all the functional elements required to promote their own secretion: an amino-terminal signal peptide to mediate translocation across the inner membrane, a central passenger domain that is the secreted functional moiety, and a channel-forming carboxyl terminus that facilitates passenger domain translocation across the outer membrane. However, recent discoveries of common structural themes, translocation intermediates and accessory interactions have challenged the perceived simplicity of autotransporter secretion. Here, we discuss how these studies have led to an improved understanding of the mechanisms responsible for autotransporter biogenesis.

The Essential β-Barrel Assembly Machinery Complex Components BamD and BamA Are Required for Autotransporter Biogenesis

Autotransporter biogenesis is dependent upon BamA, a central component of the β-barrel assembly machinery (BAM) complex. In this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter biogenesis and show that BamB, BamC, and BamE are not required.

Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins

Background: There is a general paucity of cysteine residues within the passenger domains of autotransporter proteins. Results: Distantly spaced cysteines forming disulfide-bonded loops or those enclosing structural elements are secretion-incompetent. Conclusion: Only closely spaced cysteine pairs are compatible with the autotransporter pathway. Significance: Secretion of folded peptides by the autotransporter pathway is limited; hence autotransporters lack large disulfide-bonded loops to remain secretion-competent. Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.

Transcription of the plasmid-encoded toxin gene from Enteroaggregative Escherichia coli is regulated by a novel co-activation mechanism involving CRP and Fis

Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhoea in developing countries. EAEC 042 is the prototypical strain. EAEC 042 secretes the functionally well‐characterized Pet autotransporter toxin that contributes to virulence through its cytotoxic effects on intestinal epithelial cells. Following a global transposon mutagenesis screen of EAEC 042, the transcription factors, CRP and Fis, were identified as essential for transcription of the pet gene. Using both in vivo and in vitro techniques, we show that the pet promoter is co‐dependent on CRP and Fis. We present a novel co‐activation mechanism whereby CRP is placed at a non‐optimal position for transcription initiation, creating dependence on Fis for full activation of pet. This study complements previous findings that establish Fis as a key virulence regulator in EAEC 042.

E. coli Fis Protein Insulates the cbpA Gene from Uncontrolled Transcription

The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and co-chaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual “insulation” mechanism. Thus, Fis protects cbpA from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the E. coli chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.

Mutational and topological analysis of the Escherichia coli BamA protein.

The multi-protein β-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of β-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded β-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a β-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA β-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify β-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA β-barrel are essential.

Cross‐species chimeras reveal BamA POTRA and β‐barrel domains must be fine‐tuned for efficient OMP insertion

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram‐negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β‐barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β‐barrel precursors via the five polypeptide transport‐associated (POTRA) domains at its N‐terminus. The C‐terminus of BamA folds into a β‐barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram‐negative bacteria and appear to function in a species‐specific manner. Here we investigate the nature of this species‐specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β‐barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β‐barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β‐barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.

Sequencing a piece of history:: complete genome sequence of the original Escherichia coli strain

In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5 144 392 bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2 803 932 bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577 351 bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157 : H7; therefore, it is not an ancestral state for the species.

A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization

The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as “lipid rafts.” Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

Mammalian Cell Entry domains are required for bile resistance and virulence in Salmonella

MCE domains were first reported in Mycobacteria as having a role in Mammalian Cell Entry, with subsequent studies showing their importance during infection. Here, we have examined the function of MCE proteins in Salmonella Typhimurium during mammalian infection. We report that MCE proteins are required for Salmonella virulence, but that this is not related to decreased adherence, entry or survival in mammalian cells. Instead, we reveal that MCE proteins are required for Salmonella bile resistance, in particular to withstand bile salts such as cholate and deoxycholate. Based on our previous work in Escherichia coli, and other studies that have reported roles for MCE proteins in membrane biogenesis, we propose that Salmonella lacking MCE domains have a defective outer membrane that results in bile sensitivity and decreased virulence in vivo. These results suggest that MCE domains mediate fundamental aspects of bacterial membrane physiology as opposed to a proposed direct role in mammalian cell entry, explaining their conservation across both pathogenic and non-pathogenic bacteria.