Autotransporter Protein Research Articles

Unveiling the hidden arsenal: new insights into Proteus mirabilis virulence in UTIs.

Proteus mirabilis is a Gram-negative bacterium commonly found in urinary tract infections (UTIs) and catheter-associated urinary tract infections (CAUTIs). The pathogenic mechanisms of Proteus mirabilis are complex and diverse, involving various virulence factors, including fimbriae, flagella, urease, polyphosphate kinase, lipopolysaccharides, cyclic AMP receptor protein, Sigma factor RpoE, and RNA chaperone protein Hfq. These factors play crucial roles in bacterial colonization, invasion, evasion of host immune responses, biofilm formation, and urinary stone formation. This paper is the first to comprehensively describe the hydrogenase system, autotransporter proteins, molybdate-binding protein ModA, and two-component systems as virulence factors in Proteus mirabilis, providing new insights into its pathogenic mechanisms in urinary tract infections. This review explores the mechanisms of biofilm formation by Proteus mirabilis and the various virulence factors involved in UTIs, revealing many newly discovered virulence factors from recent studies. These findings may offer new targets for clinical treatment of UTIs and vaccine development, highlighting the importance of understanding these virulence factors.

Bacterial Living Therapeutics with Engineered Protein Secretion Circuits to Eliminate Breast Cancer Cells.

Cancer therapy can be limited by potential side effects, and bacteria-based living cancer therapeutics have gained scientific interest in recent years. However, the full potential of bacteria as therapeutics has yet to be explored due to engineering challenges. In this study, we present a bacterial device designed to specifically target and eliminate breast cancer cells. We have engineered Escherichia coli (E. coli) to bind to HER2 receptors on breast cancer cells while also secreting a toxin, HlyE, which is a pore-forming protein. The binding of E. coli to HER2 is facilitated by a nanobody expressed on the bacteria's surface via the Ag43 autotransporter protein system. Our findings demonstrate that the nanobody efficiently binds to HER2+ cells in vitro, and we have utilized the YebF secretion tag to secrete HlyE and kill the target cancer cells. Overall, our results highlight the potential of our engineered bacteria as an innovative strategy for breast cancer treatment.

Genomic Dissection of an Enteroaggregative Escherichia coli Strain Isolated from Bacteremia Reveals Insights into Its Hybrid Pathogenic Potential.

Escherichia coli is a frequent pathogen isolated from bloodstream infections. This study aimed to characterize the genetic features of EC092, an E. coli strain isolated from bacteremia that harbors enteroaggregative E. coli (EAEC) genetic markers, indicating its hybrid pathogenic potential. Whole-genome sequencing showed that EC092 belongs to phylogroup B1, ST278, and serotype O165:H4. Genes encoding virulence factors such as fimbriae, toxins, iron-uptake systems, autotransporter proteins (Pet, Pic, Sat, and SepA), and secretion systems were detected, as well as EAEC virulence genes (aggR, aatA, aaiC, and aap). EC092 was found to be closely related to the other EAEC prototype strains and highly similar in terms of virulence to three EAEC strains isolated from diarrhea. The genomic neighborhood of pet, pic, sat, sepA, and the EAEC virulence genes of EC092 and its three genetically related fecal EAEC strains showed an identical genomic organization and nucleotide sequences. Also, EC092 produced and secreted Pet, Pic, Sat, and SepA in the culture supernatant and resisted the bactericidal activity of normal human serum. Our results demonstrate that the strain EC092, isolated from bacteremia, is a hybrid pathogenic extraintestinal E. coli (ExPEC)/EAEC with virulence features that could mediate both extraintestinal and intestinal infections.

The Assembly of the Inverse Autotransporter Protein YeeJ is Driven by its C-terminal β-strand

Autotransporter proteins are bacterial outer membrane proteins that display passenger domains with various functions through a β-barrel shaped translocation domain. YeeJ is an autotransporter protein from E. coli MG1655. In contrast to most other autotransporter proteins, its passenger domain is located at the C-terminus of the translocation domain. Due to this inverted domain organization, YeeJ belongs to autotransporter proteins of type Ve. To investigate the assembly of YeeJ, the fluorescence of a heterologous mCherry passenger domain was measured to quantify its assembly. Based on AlphaFold2 models of 119 sequences similar to YeeJ, a sequence conservation logo for the β1- and the β12-strand of type Ve autotransporter proteins was generated. Then, the effect of mutations in these strands on the assembly of YeeJ were analyzed. Mutations of the N-terminal aromatic amino acid of the β1-strand did not affect the assembly of the translocation domain and the display of the passenger domain. Likewise, exchange of the β1-strand with the β3-strand did not impair the assembly of the autotransporter fusion protein. Mutation of the C-terminal aromatic amino acid of the β12-strand strongly impaired surface display of the mCherry passenger domain. This amino acid has been shown before as an essential feature of the β-signals of classical autotransporter proteins and outer membrane β-barrel proteins in general. We therefore propose that the β12-strand of YeeJ acts as its β-signal and that the assembly of the YeeJ β-barrel is driven by its C-terminal β-strand, like in most other autotransporter proteins, despite its inverted domain organization.

Plasmid-encoded toxin of Escherichia coli cleaves complement system proteins and inhibits complement-mediated lysis in vitro.

Plasmid-encoded toxin (Pet) is an autotransporter protein of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, important in the pathogenicity of Escherichia coli. The pet gene was initially found in the enteroaggregative E. coli (EAEC) virulence plasmid, pAA2. Although this virulence factor was initially described in EAEC, an intestinal E. coli pathotype, pet may also be present in other pathotypes, including extraintestinal pathogenic strains (ExPEC). The complement system is an important defense mechanism of the immune system that can be activated by invading pathogens. Proteases produced by pathogenic bacteria, such as SPATEs, have proteolytic activity and can cleave components of the complement system, promoting bacterial resistance to human serum. Considering these factors, the proteolytic activity of Pet and its role in evading the complement system were investigated. Proteolytic assays were performed by incubating purified components of the complement system with Pet and Pet S260I (a catalytic site mutant) proteins. Pet, but not Pet S260I, could cleave C3, C5 and C9 components, and also inhibited the natural formation of C9 polymers. Furthermore, a dose-dependent inhibition of ZnCl2-induced C9 polymerization in vitro was observed. E. coli DH5α survived incubation with human serum pre-treated with Pet. Therefore, Pet can potentially interfere with the alternative and the terminal pathways of the complement system. In addition, by cleaving C9, Pet may inhibit membrane attack complex (MAC) formation on the bacterial outer membrane. Thus, our data are suggestive of a role of Pet in resistance of E. coli to human serum.

The ways Fusobacterium nucleatum translocate to breast tissue and contribute to breast cancer development.

Breast cancer is among the most prevalent malignancies in women worldwide. Epidemiological findings suggested that periodontal diseases may be associated with breast cancer, among which Fusobacterium nucleatum is considered an important cross-participant. In this work, we comprehensively summarize the known mechanisms of how F. nucleatum translocates to, colonizes in mammary tumors, and promotes the carcinogenesis. Specifically, F. nucleatum translocates to mammary tissue through the mammary-intestinal axis, direct nipple contact, and hematogenous transmission. Subsequently, F. nucleatum takes advantage of fusobacterium autotransporter protein 2 to colonize breast cancer and uses virulence factors fusobacterium adhesin A and lipopolysaccharide to promote proliferation. Moreover, the upregulated matrix metalloproteinase-9 induced by F. nucleatum does not only trigger the inflammatory response but also facilitates the tumor-promoting microenvironment. Aside from the pro-inflammatory effect, F. nucleatum may also be engaged in tumor immune evasion, which is achieved through the action of virulence factors on immune checkpoint receptors highly expressed on T cells, natural killer cells, and tumor-infiltrating lymphocytes. Taking breast cancer as an example, more relevant research studies may expand our current knowledge of how oral microbes affect systemic health. Hopefully, exploring these mechanisms in depth could provide new strategies for safer and more effective biologic and targeted therapies targeted at breast cancer.

Split-GFP complementation at the bacterial cell surface for antibody-free labeling and quantification of heterologous protein display

The split-GFP system is a versatile tool with numerous applications, but it has been underutilized for the labeling of heterologous surface-displayed proteins. By inserting the 16 amino acid sequence of the GFP11-tag between a protein of interest and an autotransporter protein, it is possible to present a protein at the outer membrane of gram-negative bacteria and to fluorescently label it by complementation with externally added GFP1–10. The labeled cells could be clearly discerned from cells without the protein of interest using flow cytometry and the insertion of the GFP11-tag caused no significant alteration of the catalytic activity for the tested model enzyme CsBglA. Furthermore, the amount of the protein of interest on the cells could be quantified by comparing the green fluorescence resulting from the complementation to that of standards with known concentrations. This allows a precise characterization of whole-cell biocatalysts, which is difficult with existing methods. The split-GFP complementation approach was shown to be specific, in a similar manner as commercial antibodies. It is cost-efficient, minimizes the possibility of adverse effects on protein expression or solubility, and can be performed at high throughput.

Characterization of HcaA, a novel autotransporter protein in Helicobacter cinaedi, and its role in host cell adhesion.

Helicobacter species are classified as gastric or enterohepatic according to their habitat. Among enterohepatic Helicobacter species, which inhabit the intestine, colon, and liver, Helicobacter cinaedi has been most frequently isolated from humans. H. cinaedi often causes bacteremia and cellulitis in immunocompromised hosts. Here, we focused on the H. cinaedi autotransporter protein A (HcaA), a novel virulence factor in H. cinaedi. We discovered that HcaA contributes to cell adhesion via its Arg-Gly-Asp motif. Furthermore, in animal experiments, bacterial colonization was reduced in mice infected with HcaA-knockout strains, supporting the hypothesis that HcaA contributes to H. cinaedi adhesion to host cells. Our study provides a novel mechanism for the establishment of H. cinaedi infections and provides new insights into the role of autotransporter proteins in the establishment of Helicobacter infection.

A structural foundation for studying chlamydial polymorphic membrane proteins.

Polymorphic membrane proteins (Pmps) are chlamydial cell surface proteins with signature repeating FxxN and GGA (I, V, L) tetrapeptide motifs. These autotransporter proteins are key immune targets and mediators of host-microbe interactions but by unknown mechanisms. AlphaFold predictions reveal that Pmp passenger domains have two distinct β-helical parts: a C-terminal globular "Pmp middle region" that forms six β-helical rungs, and an N-terminal highly-regular "Pmp repeat region" that is from 9 to 25 rungs long. In the repeat region, paired FxxN and GGA (I, V, L) motifs - or close variants - are fully buried and packed together to form novel β-helical rungs from which variable-length loops extend out from one face. Similar motifs in proteins from diverse organisms are predicted to form analogous structures. In Pmps, the structures imply that the tetrapeptide motifs are not directly involved in host-cell adhesion, but position protein loops on a common face where they can interact with ligands important for infection. These structural descriptions provide a guide for mutagenesis studies to identify those parts of Pmps involved in infection.IMPORTANCEInfections by bacteria in the genus Chlamydia cause a range of widespread and potentially debilitating conditions in humans and other animals. We analyzed predicted structures of a family of proteins that are potential vaccine targets found in all Chlamydia spp. Our findings deepen the understanding of protein structure, provide a descriptive framework for discussion of the protein structure, and outline regions of the proteins that may be key targets in host-microbe interactions and anti-chlamydial immunity.

Phylogenetic, structural and functional studies of bacterial autotransporter proteins reveals novel approaches for drug development and delivery

Phylogenetic, structural and functional studies of bacterial autotransporter proteins reveals novel approaches for drug development and delivery

Sialic acid plays a pivotal role in licensing Citrobacter rodentium’s transition from the intestinal lumen to a mucosal adherent niche

Enteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses and an established microbiota remain poorly defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy likely involves metabolic adaptation to the host's intestinal luminal environment, as a necessary precursor to reach and infect the mucosal surface. Suspecting this adaptation involved the intestinal mucus layer, we found that C. rodentium was able to catabolize sialic acid, a monosaccharide derived from mucins, and utilize it as its sole carbon source for growth. Moreover, C. rodentium also sensed and displayed chemotactic activity toward sialic acid. These activities were abolished when the nanT gene, encoding a sialic acid transporter, was deleted (ΔnanT). Correspondingly, the ΔnanT C. rodentium strain was significantly impaired in its ability to colonize the murine intestine. Intriguingly, sialic acid was also found to induce the secretion of two autotransporter proteins, Pic and EspC, which possess mucinolytic and host-adherent properties. As a result, sialic acid enhanced the ability of C. rodentium to degrade intestinal mucus (through Pic), as well as to adhere to intestinal epithelial cells (through EspC). We thus demonstrate that sialic acid, a monosaccharide constituent of the intestinal mucus layer, functions as an important nutrient and a key signal for an A/E bacterial pathogen to escape the colonic lumen and directly infect its host's intestinal mucosa.

Can bacteria F. nucleatum be actively involved in colon cancer progression via a radical mediated mechanism?

Outer membrane proteins of Fusobacterium nucleatum, a cancer‑leading bacteria, are considered as the factors responsible for its pathogenicity. Among them, homotrimeric autotransporter protein YadA (Yersinia adhesin A) is an important virulence factor also found in the outer membrane of pathogenic Yersinia species. In this paper, the structure and stability of certain Cu(II) complexes with YadA fragments were investigated using both, experimental and theoretical methods. Potentiometry, UV–Vis, CD, EPR, and calculations at the density functional theory (DFT) level were applied to determine the metal ion coordination sphere. Moreover, the complexes ability to DNA cleavage and reactive oxygen species (ROS) production was studied. We have shown that copper(II) complexes can cleave DNA by 1O2, O2•− and •OH, which are formed in the studied systems. However, the results of electrophoretic experiments revealed that complexes cleave DNA less effectively than free copper(II) ions. Therefore, the presence of studied peptides may prevent DNA from a Cu(II)-induced damage to some extent.

Identification of endometrial biofilm-specific proteins for in vivo diagnostics

Bacteria enter the uterus in a free-floating planktonic state, which makes them vulnerable to the host-immune system as well as exposure to effective antimicrobials. If not eliminated in a timely fashion, the bacteria will adhere to the endometrial surface and may form micro-colonies, develop a complex matrix, and switch into a biofilm community. The ability of bacteria to shift from planktonic to biofilm phenotypes has been described for both Gram-negative and Gram-positive bacteria in human mucosal tissue, indwelling catheters, and implants as well as other organs, including the equine endometrium. Unfortunately, there is currently no available assay to diagnose the condition in vivo. Literature in human medicine suggests that biofilm matrices contain both secretory and non-secretory proteins, and while some of the proteins are common among all biofilms, others may be specific for bacterial species. Therefore, the objective of this research was to identify specific proteins from biofilm-producing bacteria from mares with endometritis. Whole-cell extracts from potentially biofilm-forming bacterial isolates of Streptococcus zooepidemicus (SEZ) (n=3), Pseudomonas aeruginosa (n=1), and Escherichia coli (n=4) were analyzed by 2-D PAGE. Differentially expressed proteins were selected by overlapping 2-D PAGE biofilm proteins with planktonic culture proteins.In-gel analysis of differences (with a 3-fold cut-off)identified a total of 96 proteins for SEZ, 83 proteins for P. aeruginosa, and 69 proteins for E. coli as potential biofilm markers. Select proteins were further identified by MALDI-TOF/TOF. In SEZ, glutathione-disulfide reductase (48.8 kDa, PI 5.6), which catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant, and TIGR00266 (25.3 kDa, PI 5.4) were identified as proteins of interest. For P. aeruginosa, thioredoxin A (11.8 kDa, PI 4.7), which participates in various redox reactions, osmotically inducible protein C (15.6 kDa, PI 5.9), important for cellular defense mechanism against oxidative stress, and bacterial extracellular solute-binding s, 3 family protein (33 kDa, PI 8.3) serve as potential targets. Potential biomarker candidates for E. coli include the serine protease pet autotransporter (139 kDa, PI 6.5) and outer membrane protein X (18 kDa, PI 6.6), a protein involved in numerous physiological processes including invasion, signal transduction, and binding external proteins. In conclusion, we identified biofilm-specific proteins that may serve as in vivo diagnostic markers for biofilms in uterine lavage from mares with biofilm-associated bacterial endometritis. Future research will pursue these proteins of interest for their use as diagnostic tools in the field.

Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli.

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

Single particle cryo-EM analysis of Rickettsia conorii Sca2 reveals a formin-like core

Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an ∼ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first ∼ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.

Broad protective vaccination against systemic Escherichia coli with autotransporter antigens.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of adult life-threatening sepsis and urinary tract infections (UTI). The emergence and spread of multidrug-resistant (MDR) ExPEC strains result in a considerable amount of treatment failure and hospitalization costs, and contribute to the spread of drug resistance amongst the human microbiome. Thus, an effective vaccine against ExPEC would reduce morbidity and mortality and possibly decrease carriage in healthy or diseased populations. A comparative genomic analysis demonstrated a gene encoding an invasin-like protein, termed sinH, annotated as an autotransporter protein, shows high prevalence in various invasive ExPEC phylogroups, especially those associated with systemic bacteremia and UTI. Here, we evaluated the protective efficacy and immunogenicity of a recombinant SinH-based vaccine consisting of either domain-3 or domains-1,2, and 3 of the putative extracellular region of surface-localized SinH. Immunization of a murine host with SinH-based antigens elicited significant protection against various strains of the pandemic ExPEC sequence type 131 (ST131) as well as multiple sequence types in two distinct models of infection (colonization and bacteremia). SinH immunization also provided significant protection against ExPEC colonization in the bladder in an acute UTI model. Immunized cohorts produced significantly higher levels of vaccine-specific serum IgG and urinary IgG and IgA, findings consistent with mucosal protection. Collectively, these results demonstrate that autotransporter antigens such as SinH may constitute promising ExPEC phylogroup-specific and sequence-type effective vaccine targets that reduce E. coli colonization and virulence.

Investigating the sequence properties that govern disorder and secretion of bacterial virulence proteins.

Many proteins contain disordered segments that play important roles in vivo. Autotransporter (AT) proteins are key virulence proteins from pathogenic Gram-negative bacteria. AT proteins have disordered properties that have been shown to be crucial for their efficient secretion to the bacterial cell surface. We have extensively studied pertactin, a virulence protein secreted by Bordetella pertussis, the bacterium that causes whooping cough. Like several other ATs, the mature pertactin virulence protein contains two segments with distinctly different folding properties. In isolation, the more stable C-terminal segment (PCt) is stably folded whereas the N-terminal segment (PNt) is disordered. Our previous studies indicate that folding of PCt is important for the efficient secretion of pertactin. Moreover, we recently showed that local clustering of hydrophobic residues leads to a significant collapse of PNt in vitro. Interestingly, we found that the extent to which PNt adopts a disordered, highly expanded conformational ensemble regulates the efficient secretion of pertactin through the bacterial outer membrane. Taken together, these observations suggest that PNt contains sequence properties important for secretion. Because disordered properties are important for AT secretion, understanding the connections between amino acid sequence features, disordered conformations, and AT secretion will improve our understanding of virulence protein secretion. The present research project aims to build upon our recent findings to develop a predictive understanding of the sequence factors that regulate disorder in pertactin and other proteins. We are using a combination of biophysical and biochemical approaches to characterize the sequence determinants of disorder and test their impact on AT secretion. We expect that insight into the regulation of disorder in pertactin will likely also apply to other members of the AT protein family, the largest family of secreted virulence proteins in Gram-negative bacterial pathogens.

Serotype-Specific Sugars Impact Structure but Not Functions of the Trimeric Autotransporter Adhesin EmaA of Aggregatibacter actinomycetemcomitans.

The human oral pathobiont Aggregatibacter actinomycetemcomitans expresses multiple virulence factors, including the trimeric, extracellular matrix protein adhesin A (EmaA). The posttranslational modification of EmaA is proposed to be dependent on the sugars and enzymes associated with O-polysaccharide (O-PS) synthesis of the lipopolysaccharide (LPS). This modification is important for the structure and function of this adhesin. To determine if the composition of the sugars alters structure and/or function, the prototypic 202-kDa protein was expressed in a non-serotype b, emaA mutant strain. The transformed strain displayed EmaA adhesins similar in appearance to the prototypic adhesin as observed by two-dimensional (2D) electron microscopy of whole-mount negatively stained bacterial preparations. Biochemical analysis indicated that the protein monomers were posttranslationally modified. 3D electron tomographic reconstruction and structure analyses of the functional domain revealed three well-defined subdomains (SI, SII, and SIII) with a linker region between SII and SIII. Structural changes were observed in all three subdomains and the linker region of the adhesins synthesized compared with the known structure. These changes, however, did not affect the ability of the strain to bind collagen or form biofilms. The data suggest that changes in the composition of the glycan moiety alter the 3D structure of the molecule without negatively affecting the function(s) associated with this adhesin. IMPORTANCE The human oral pathogen A. actinomycetemcomitans is a causative agent of periodontal and several systemic diseases. EmaA is a trimeric autotransporter protein adhesin important for colonization by this pathobiont in vivo. This adhesin is modified with sugars associated with the O-polysaccharide (O-PS), and the modification is mediated using the enzymes involved in lipopolysaccharide (LPS) biosynthesis. The interaction with collagen is not mediated by the specific binding between the glycans and collagen but is attributed to changes in the final quaternary structure necessary to maintain an active adhesin. In this study, we have determined that the composition of the sugars utilized in the posttranslational modification of this adhesin is exchangeable without compromising functional activities.

A Vaccine for Canine Rocky Mountain Spotted Fever: An Unmet One Health Need.

Outbreaks of life-threatening Rocky Mountain spotted fever in humans and dogs associated with a canine-tick maintenance cycle constitute an important One Health opportunity. The reality of the problem has been observed strikingly in Mexico, Brazil, Colombia, and Native American tribal lands in Arizona. The brown dog tick, Rhipicephalus sanguineus sensu lato, acquires the rickettsia from bacteremic dogs and can maintain the bacterium transtadially to the next tick stage. The subsequent adult tick can then transmit infection to a new host, as shown by guinea pig models. These brown dog ticks maintain spotted fever group rickettsiae transovarially through many generations, thus serving as both vector and reservoir. Vaccine containing whole-killed R. rickettsii does not stimulate sufficient immunity. Studies of Rickettsia subunit antigens have demonstrated that conformationally preserved outer-membrane autotransporter proteins A and B are the leading vaccine candidates. The possibility of a potentially safe and effective live attenuated vaccine has only begun to be explored as gene knockout methods are applied to these obligately intracellular pathogens.

Phylogenetic Classification and Functional Review of Autotransporters.

Autotransporters are the core component of a molecular nano-machine that delivers cargo proteins across the outer membrane of Gram-negative bacteria. Part of the type V secretion system, this large family of proteins play a central role in controlling bacterial interactions with their environment by promoting adhesion to surfaces, biofilm formation, host colonization and invasion as well as cytotoxicity and immunomodulation. As such, autotransporters are key facilitators of fitness and pathogenesis and enable co-operation or competition with other bacteria. Recent years have witnessed a dramatic increase in the number of autotransporter sequences reported and a steady rise in functional studies, which further link these proteins to multiple virulence phenotypes. In this review we provide an overview of our current knowledge on classical autotransporter proteins, the archetype of this protein superfamily. We also carry out a phylogenetic analysis of their functional domains and present a new classification system for this exquisitely diverse group of bacterial proteins. The sixteen phylogenetic divisions identified establish sensible relationships between well characterized autotransporters and inform structural and functional predictions of uncharacterized proteins, which may guide future research aimed at addressing multiple unanswered aspects in this group of therapeutically important bacterial factors.