Rickettsia rickettsii virulence determinants RARP2 and RapL mitigate IFN-ß signaling in primary human dermal microvascular endothelial cells.

We compared the growth characteristics of a virulent Rickettsia rickettsii strain (Sheila Smith) to an attenuated R. rickettsii stain (Iowa) and a non-pathogenic species (R. montanensis) in primary human dermal microvascular endothelial cells (HDMEC). All replicated in Vero cells, however, only the Sheila Smith strain productively replicated in HDMECs. The Iowa strain showed minimal replication over a 24-h period, while R. montanensis lost viability and induced lysis of the HDMECs via a rapid programmed cell death response. Both the virulent and attenuated R. rickettsii strains, but not R. montanensis, induced an interferon-1 response, although the response was of lesser magnitude and delayed in the Sheila Smith strain. IFN-ß secretion correlated with increased host cell lysis, and treatment with anti-IFNAR2 antibody decreased lysis from Iowa-infected but not Sheila Smith-infected cells. Both Sheila Smith- and Iowa-infected cells eventually lysed, although the response from Sheila Smith was delayed and showed characteristics of apoptosis. We, therefore, examined whether reconstitution of the Iowa strain with two recently described putative virulence determinants might enhance survival of Iowa within HDMECs. Reconstitution with RARP2, which is inhibitory to anterograde trafficking through the Golgi apparatus, reduced IFN-ß secretion but had no effect on cell lysis. RapL, which proteolytically processes surface exposed autotransporters and enhances replication of Iowa in Guinea pigs, suppressed both IFN-ß production and host cell lysis. These findings suggest distinct mechanisms by which virulent spotted fever group rickettsiae may enhance intracellular survival and replication.IMPORTANCEWe examined a naturally occurring non-pathogenic rickettsial species, R. montanensis, a laboratory-attenuated R. rickettsii strain (Iowa), and a fully virulent R. rickettsii strain (Sheila Smith) for growth in human dermal microvascular endothelial cells. The two avirulent strains replicated poorly or not at all. Only the virulent Sheila Smith strain replicated. IFN-ß production correlated with the inhibition of R. rickettsii Iowa. Reconstitution of Iowa with either of two recently described putative virulence determinants altered the IFN-ß response. A rickettsial ankyrin repeat protein, RARP2, disrupts the trans-Golgi network and inhibits IFN-ß secretion. An autotransporter peptidase, RapL, restores proteolytic maturation of outer membrane autotransporters and diminishes the IFN-ß response to enhance cell survival and permit replication of the recombinant strain. These studies point the way toward discovery of mechanisms for innate immune response avoidance by virulent rickettsia.

Surface expression of carbonic anhydrase on E. coli as a sustainable approach for enzymatic CO2 capture.

The utilisation of carbonic anhydrase (CA) in CO2 sequestration is becoming prominent as an efficient, environment friendly and rapid catalyst for capturing CO2 from industrial emissions. However, the application of CA enzyme in soluble form is constrained due to its poor stability in operational conditions of CO2 capture and also production cost of the enzyme. Addressing these limitations, the present study focuses on the surface display of CA from Bacillus halodurans (BhCA) on E coli aiming to contribute to the cost-effectiveness of carbon capture through CA technology. This involved the fusion of the BhCA-encoding gene with the adhesion molecule involved in diffuse adherence (AIDA-I) autotransporter, resulting in the efficient display of BhCA (595 ± 60 U/gram dry cell weight). Verification of the surface display of BhCA was accomplished by conjugating with FITC labelled anti-his antibody followed by fluorescence-activated cell sorting (FACS) and cellular fractionation in conjunction with zymography. Biochemical characterisation of whole-cell biocatalyst revealed a noteworthy enhancement in thermostability, improvement in the thermostability with T1/2 of 90 ± 1.52 minutes at 50 ˚C, 36 ± 2.51 minutes at 60 ˚C and18 ± 1.52 minutes at 80˚C. Surface displayed BhCA displayed remarkable reusability retaining 100% activity even after 15 cycles. Surface displayed BhCA displayed highly alkali stable nature like free counterpart in solution. The alkali stability of the surface-displayed BhCA was comparable to its free counterpart in solution. Furthermore, the study investigated the impact of different metal ions, modulators, and detergents on the whole-cell biocatalysts. The present work represents the first report on surface display of CA utilising the AIDA-1 autotransporter.

Investigation of an AIDA-I based expression system for display of various affinity proteins on Escherichia coli.

Autotransporters constitute a large family of natural proteins that are essential for delivering many types of proteins and peptides across the outer membrane in Gram-negative bacteria. In biotechnology, autotransporters have been explored for display of recombinant proteins and peptides on the surface of Escherichia coli and have potential as tools for directed evolution of affinity proteins. Here, we investigate conditions for AIDA-I autotransporter-mediated display of recombinant proteins. A new expression vector was designed and engineered for this purpose, and a panel of proteins from different affinity-protein classes were subcloned to the vector, followed by evaluation of expression, surface display and functionality. Surface expression was explored in ten different E. coli strains together with assessment of transformation efficiencies. Furthermore, the most promising strain and expression vector combination was used in mock library selections for evaluation of magnetic-assisted cell sortings (MACS). The results demonstrated dramatically different performances depending on the type of affinity protein and choice of E. coli strain. The optimized MACS protocol showed efficient enrichment, and thus potential for the new AIDA-I display system to be used in methods for directed evolution of affinity proteins.

Display of Oligo-α-1,6-Glycosidase from Exiguobacterium sibiricum on the Surface of Escherichia coli Cells.

Cell-surface display using anchor motifs of outer membrane proteins allows exposure of target peptides and proteins on the surface of microbial cells. Previously, we obtained and characterized highly catalytically active recombinant oligo-α-1,6-glycosidase from the psychrotrophic bacterium Exiguobacterium sibiricum (EsOgl). It was also shown that the autotransporter AT877 from Psychrobacter cryohalolentis and its deletion variants efficiently displayed type III fibronectin (10Fn3) domain 10 on the surface of Escherichia coli cells. The aim of the work was to obtain an AT877-based system for displaying EsOgl on the surface of bacterial cells. The genes for the hybrid autotransporter EsOgl877 and its deletion mutants EsOgl877Δ239 and EsOgl877Δ310 were constructed, and the enzymatic activity of EsOgl877 was investigated. Cells expressing this protein retained ~90% of the enzyme maximum activity within a temperature range of 15-35°C. The activity of cells expressing EsOgl877Δ239 and EsOgl877Δ310 was 2.7 and 2.4 times higher, respectively, than of the cells expressing the full-size AT. Treatment of cells expressing EsOgl877 deletion variants with proteinase K showed that the passenger domain localized to the cell surface. These results can be used for further optimization of display systems expressing oligo-α-1,6-glycosidase and other heterologous proteins on the surface of E. coli cells.

The regulation of bacterial two-partner secretion systems.

Two-partner secretion (TPS) systems, also known as Type Vb secretion systems, allow the translocation of effector proteins across the outer membrane of Gram-negative bacteria. By secreting different classes of effectors, including cytolysins and adhesins, TPS systems play important roles in bacterial pathogenesis and host interactions. Here, we review the current knowledge on TPS systems regulation and highlight specific and common regulatory mechanisms across TPS functional classes. We discuss in detail the specific regulatory networks identified in various bacterial species and emphasize the importance of understanding the context-dependent regulation of TPS systems. Several regulatory cues reflecting host environment during infection, such as temperature and iron availability, are common determinants of expression for TPS systems, even across relatively distant species. These common regulatory pathways often affect TPS systems across subfamilies with different effector functions, representing conserved global infection-related regulatory mechanisms.

Quantitative Proteomic Analysis of Outer Membrane Vesicles from Fusobacterium nucleatum Cultivated in the Mimic Cancer Environment.

Fusobacterium nucleatum is a Gram-negative bacterium that has been identified as an important pathogenic gut bacterium associated with colorectal cancer. Compared with the normal intestine, the pH value of the tumor microenvironment is weakly acidic. The metabolic changes of F. nucleatum in the tumor microenvironment, especially the protein composition of its outer membrane vesicles, remain unclear. Here, we systematically analyzed the effect of environmental pH on the proteome of outer membrane vesicles (OMVs) from F. nucleatum by tandem mass tag (TMT) labeling-high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 991 proteins were identified in acidic OMVs (aOMVs) and neutral OMVs (nOMVs), including known virulence proteins and putative virulence proteins. Finally, 306 upregulated proteins and 360 downregulated proteins were detected in aOMVs, and approximately 70% of the expression of OMV proteins was altered under acidic conditions. A total of 29 autotransporters were identified in F. nucleatum OMVs, and 13 autotransporters were upregulated in aOMVs. Interestingly, three upregulated autotransporters (D5REI9, D5RD69, and D5RBW2) show homology to the known virulence factor Fap2, suggesting that they may be involved in various pathogenic pathways such as the pathway for binding with colorectal cancer cells. Moreover, we found that more than 70% of MORN2 domain-containing proteins may have toxic effects on host cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that a number of proteins were significantly enriched in multiple pathways involving fatty acid synthesis and butyrate synthesis. Seven metabolic enzymes involved in fatty acid metabolism pathways were identified in the proteomic data, of which 5 were upregulated and 2 were downregulated in aOMVs, while 14 metabolic enzymes involved in the butyric acid metabolic pathway were downregulated in aOMVs. In conclusion, we found a key difference in virulence proteins and pathways in the outer membrane vesicles of F. nucleatum between the tumor microenvironment pH and normal intestinal pH, which provides new clues for the prevention and treatment of colorectal cancer. IMPORTANCE F. nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. OMVs have been demonstrated to play key roles in pathogenesis by delivering toxins and other virulence factors to host cells. By employing quantitative proteomic analysis, we found that the pH conditions could affect the protein expression of the outer membrane vesicles of F. nucleatum. Under acidic conditions, approximately 70% of the expression of proteins in OMVs was altered. Several virulence factors, such as type 5a secreted autotransporter (T5aSSs) and membrane occupation and recognition nexus (MORN) domain-containing proteins, were upregulated under acidic conditions. A large number of proteins showed significant enrichments in multiple pathways involving fatty acid synthesis and butyrate synthesis. Proteomics analysis of the outer membrane vesicles secreted by pathogenic bacteria in the acidic tumor microenvironment is of great significance for elucidating the pathogenicity mechanism and its application in vaccine and drug delivery vehicles.

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.

Comparison of Biological Activities of BafA Family Autotransporters within Bartonella Species Derived from Cats and Rodents.

Bartonella species are hemotropic, facultative intracellular bacteria, some of which cause zoonoses, that are widely disseminated among many mammals, including humans. During infection in humans, vascular endothelial cells play a crucial role as a replicative niche for Bartonella, and some are capable of promoting vascular proliferation. Along with well-studied pathogenic factors such as a trimeric autotransporter adhesin BadA or VirB/D4 type IV secretion system, bacteria-secreted protein BafA is also involved in Bartonella-induced vasoproliferation. Genes encoding BafA orthologs have been found in the genomes of most Bartonella species, but their functionality remains unclear. In this study, we focused on three cat-derived zoonotic species (B. henselae, B. koehlerae, and B. clarridgeiae) and two rodent-derived species (B. grahamii and B. doshiae) and compared the activity of BafA derived from each species. Recombinant BafA proteins of B. henselae, B. koehlerae, B. clarridgeiae, and B. grahamii, species that also cause human disease, induced cell proliferation and tube formation in cultured endothelial cells, while BafA derived from B. doshiae, a species that is rarely found in humans, showed neither activity. Additionally, treatment of cells with these BafA proteins increased phosphorylation of both vascular endothelial growth factor receptor 2 and extracellular signal-regulated kinase 1/2, with the exception of B. doshiae BafA. Differential bafA mRNA expression and BafA secretion among the species likely contributed to the differences in the cell proliferation phenotype of the bacteria-infected cells. These findings suggest that the biological activity of BafA may be involved in the infectivity or pathogenicity of Bartonella species in humans.

An Alternative Splicing Variant of the Mixed-Lineage Leukemia 5 Protein Is a Cellular Adhesion Receptor for ScaA of Orientia tsutsugamushi.

Scrub typhus is a mite-borne disease caused by the obligately intracellular bacterium Orientia tsutsugamushi. We previously demonstrated that ScaA, an autotransporter membrane protein of O. tsutsugamushi, is commonly shared in various genotypes and involved in adherence to host cells. Here, we identified a mixed-lineage leukemia 5 (MLL5) mammalian trithorax group protein as a host receptor that interacts with ScaA. MLL5, identified by yeast two-hybrid screening, is an alternative splicing variant of MLL5 (vMLL5) which contains 13 exons with additional intron sequences encoding a tentative transmembrane domain. Indeed, vMLL5 is expressed on the plasma membrane as well as in intracellular compartments in eukaryotic cells and colocalized with adherent O. tsutsugamushi. In addition, ScaA-expressing Escherichia coli showed significantly increased adherence to vMLL5-overexpressing cells compared with vector control cells. We mapped the C-terminal region of the passenger domain of ScaA as a ligand for vMLL5 and determined that the Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain of MLL5 is an essential and sufficient motif for ScaA binding. We observed significant and specific inhibition of bacterial adhesion to host cells in competitive inhibition assays using the C-terminal fragment of ScaA or the SET domain of vMLL5. Moreover, immunization with the C-terminal fragment of ScaA provided neutralizing activity and protective immunity against lethal challenge with O. tsutsugamushi as efficiently as vaccination with the whole passenger domain of ScaA. These results indicate that vMLL5 is a novel cellular receptor for ScaA-mediated adhesion of O. tsutsugamushi and facilitates bacterial adhesion to host cells, thereby enhancing bacterial infection. IMPORTANCE O. tsutsugamushi is a mite-borne pathogen that causes scrub typhus. As an obligately intracellular pathogen, its adhesion to and invasion of host cells are critical steps for bacterial growth. However, the molecular basis of the bacterial ligand and host receptor interaction is poorly defined. Here, we identified a splicing variant of MLL5 (vMLL5) as a cellular adhesion receptor of ScaA, an outer membrane autotransporter protein of O. tsutsugamushi. We mapped the interacting domains in the bacterial ligand and host receptor and confirmed their functional interaction. In addition, immunization with the C-terminal region of ScaA, which involves an interaction with the SET domain of vMLL5, not only induces enhanced neutralizing antibodies but also provides protective immunity against lethal challenge with O. tsutsugamushi.

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.

Cysteine-Dependent Conformational Heterogeneity of Shigella flexneri Autotransporter IcsA and Implications of Its Function.

Shigella IcsA is a versatile surface virulence factor required for early and late pathogenesis stages extracellularly and intracellularly. Despite IcsA serving as a model Type V secretion system (T5SS) autotransporter to study host-pathogen interactions, its detailed molecular architecture is poorly understood. Recently, IcsA was found to switch to a different conformation for its adhesin activity upon sensing the host stimuli by Shigella Type III secretion system (T3SS). Here, we reported that the single cysteine residue (C130) near the N terminus of the IcsA passenger had a role in IcsA adhesin activity. We also showed that the IcsA passenger (IcsAp) existed in multiple conformations, and the conformation populations were influenced by a central pair of cysteine residues (C375 and C379), which was not previously reported for any Type V autotransporter passengers. Disruption of either or both central cysteine residues altered the exposure of IcsA epitopes to polyclonal anti-IcsA antibodies previously shown to block Shigella adherence, yet without loss of IcsA intracellular functions in actin-based motility (ABM). Anti-IcsA antibody reactivity was restored when the IcsA-paired cysteine substitution mutants were expressed in an ΔipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovered a novel molecular switch empowered by a centrally localized, short-spaced cysteine pair in the Type V autotransporter IcsA that ensured conformational heterogeneity to aid IcsA evasion of host immunity. IMPORTANCE Shigella species are the leading cause of diarrheal-related death globally by causing bacillary dysentery. The surface virulence factor IcsA, which is essential for Shigella pathogenesis, is a unique multifunctional autotransporter that is responsible for cell adhesion, and actin-based motility, yet detailed mechanistic understanding is lacking. Here, we showed that the three cysteine residues in IcsA contributed to the protein's distinct functions. The N-terminal cysteine residue within the IcsA passenger domain played a role in adhesin function, while a centrally localized cysteine pair provided conformational heterogeneity that resulted in IcsA molecules with different reactivity to adhesion-blocking anti-IcsA antibodies. In synergy with the Type III secretion system, this molecular switch preserved biological function in distinct IcsA conformations for cell adhesion, actin-based motility, and autophagy escape, providing a potential strategy by which Shigella evades host immunity and targets this essential virulence factor.

FHUSPA2/10 is a bactericidal monoclonal antibody targeting multiple repeated sequences of Moraxella catarrhalis UspA2.

Moraxella catarrhalis is an important and common respiratory pathogen that can cause Otitis Media, Community Acquired Pneumonia, and has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease in adults, leading to morbidity and mortality. Its ubiquitous surface protein A2 (UspA2) has been shown to interact with host structures and extracellular matrix proteins, suggesting a role at an early stage of infection and a contribution to bacterial serum resistance. The UspA proteins are homo-trimeric autotransporters that appear as a lollipop-shaped structure in electron micrographs. They are composed of an N-terminal head with adhesive properties, followed by a stalk, which ends by an amphipathic helix and a C-terminal membrane domain. The three family members UspA1, UspA2 and UspA2H, present different amino acid signatures both at the head and membrane-spanning regions. By combining electron microscopy, hydrogen deuterium exchange mass spectrometry and protein modeling, we identified a shared and repeated epitope recognized by FHUSPA2/10, a potent cross-bactericidal monoclonal antibody raised by UspA2 and deduced key amino acids involved in the binding. The finding strengthens the potential of UspA2 to be incorporated in a vaccine formulation against M. catarrhalis.

Deletion Variants of Autotransporter from Psychrobacter cryohalolentis Increase Efficiency of 10FN3 Exposure on the Surface of Escherichia coli Cells.

The autotransporter AT877 from Psychrobacter cryohalolentis belongs to the family of outer membrane proteins containing N-terminal passenger and C-terminal translocator domains that form the basis for the design of display systems on the surface of bacterial cells. It was shown in our previous study that the passenger domain of AT877 can be replaced by the cold-active esterase EstPc or the tenth domain of fibronectin type III (10Fn3). In order to increase efficiency of the 10Fn3 surface display in Escherichia coli cells, four deletion variants of the Fn877 hybrid autotransporter were obtained. It was demonstrated that all variants are present in the membrane of bacterial cells and facilitate binding of the antibodies specific against 10Fn3 on the cell surface. The highest level of binding is provided by the variants Δ239 and Δ310, containing four and seven beta-strands out of twelve that comprise the structure of the translocator domain. Using electrophoresis under semi-native conditions, presence of heat modifiability in the full-size Fn877 and its deletion variants was demonstrated, which indicated preservation of beta structure in their molecules. The obtained results could be used to optimize the bacterial display systems of 10Fn3, as well as of other heterologous passenger domains.

Adhesion of Bartonella henselae to Fibronectin Is Mediated via Repetitive Motifs Present in the Stalk of Bartonella Adhesin A.

Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram-negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). Bartonella henselae targets the extracellular matrix glycoprotein fibronectin (Fn) via the Bartonella adhesin A (BadA) attaching the bacteria to the host cell. The TAA BadA is characterized by a highly repetitive passenger domain consisting of 30 neck/stalk domains with various degrees of similarity. To elucidate the motif sequences mediating Fn binding, we generated 10 modified BadA constructs and verified their expression via Western blotting, confocal laser scanning, and electron microscopy. We analyzed their ability to bind human plasma Fn using quantitative whole-cell enzyme-linked immunosorbent assays (ELISAs) and fluorescence microscopy. Polyclonal antibodies targeting a 15-mer amino acid motif sequence proved to reduce Fn binding. We suggest that BadA adheres to Fn in a cumulative effort with quick saturation primarily via unpaired ß-strands appearing in motifs repeatedly present throughout the neck/stalk region. In addition, we demonstrated that the length of truncated BadA constructs correlates with the immunoreactivity of human patient sera. The identification of BadA-Fn binding regions will support the development of new "antiadhesive" compounds inhibiting the initial adherence of B. henselae and other TAA-expressing pathogens to host cells. IMPORTANCE Trimeric autotransporter adhesins (TAAs) are important virulence factors and are widely present in various pathogenic Gram-negative bacteria. TAA-expressing bacteria cause a wide spectrum of human diseases, such as cat scratch disease (Bartonella henselae), enterocolitis (Yersinia enterocolitica), meningitis (Neisseria meningitis), and bloodstream infections (multidrug-resistant Acinetobacter baumannii). TAA-targeted antiadhesive strategies (against, e.g., Bartonella adhesin A [BadA], Yersinia adhesin A [YadA], Neisseria adhesin A [NadA], and Acinetobacter trimeric autotransporter [Ata]) might represent a universal strategy to counteract such bacterial infections. BadA is one of the best characterized TAAs, and because of its high number of (sub)domains, it serves as an attractive adhesin to study the domain-function relationship of TAAs in the infection process. The identification of common binding motifs between TAAs (here, BadA) and their major binding partner (here, fibronectin) provides a basis toward the design of novel "antiadhesive" compounds preventing the initial adherence of Gram-negative bacteria in infections.

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.

The XadA Trimeric Autotransporter Adhesins in Xylella fastidiosa Differentially Contribute to Cell Aggregation, Biofilm Formation, Insect Transmission and Virulence to Plants.

Surface adhesion strategies are widely employed by bacterial pathogens during establishment and systemic spread in their host. A variety of cell-surface appendages such as pili, fimbriae, and afimbrial adhesins are involved in these processes. The phytopathogen Xylella fastidiosa employs several of these structures for efficient colonization of its insect and plant hosts. Among the adhesins encoded in the X. fastidiosa genome, three afimbrial adhesins, XadA1, Hsf/XadA2, and XadA3, are predicted to be trimeric autotransporters with a C-terminal YadA-anchor membrane domain. We analyzed the individual contributions of XadA1, XadA2, and XadA3 to various cellular behaviors both in vitro and in vivo. Using isogenic X. fastidiosa mutants, we found that cell-cell aggregation and biofilm formation were severely impaired in the absence of XadA3. No significant reduction of cell-surface attachment was found with any mutant under flow conditions. Acquisition by insect vectors and transmission to grapevines were reduced in the XadA3 deletion mutant. While the XadA3 mutant was hypervirulent in grapevines, XadA1 or XadA2 deletion mutants conferred lower disease severity than the wild-type strain. This insight of the importance of these adhesive proteins and their individual contributions to different aspects of X. fastidiosa biology should guide new approaches to reduce pathogen transmission and disease development. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Short Peptide of Autotransporter Ata Is a Promising Protective Antigen for Vaccination Against Acinetobacter baumannii.

With the emergence of multidrug-resistant strains, Acinetobacter baumannii infection is becoming a thorny health problem in hospitals. However, there are no licensed vaccines against A. baumannii. Acinetobacter trimeric autotransporter (Ata) is an important known virulence factor located on the outer membrane of bacteria. Herein, we carried out a series of experiments to test the immunogenicity of a short C-terminal extracellular region of Ata (Ataα, only containing 39 amino acids) in a murine model. The short peptide Ataα was fused with the cholera toxin B subunit (CTB), which has been reported to have immunoadjuvant activity. The fusion protein showed no inflammation and organ damages, and have the ability to elicit both Th1 and Th2 immune responses in mice. The bactericidal activities against A. baumannii and prophylactic effects of the fusion protein were further evidenced by a significant reduction in the bacterial load in the organs and blood. In addition, the candidate vaccine could provide broad protection against lethal challenges with a variety of A. baumannii strains. Moreover, when CpG was added on the basis of aluminum adjuvant, the immune response, especially cellular immunity, could be further strengthened. Overall, these results revealed that the Ataα is a promising vaccine target against A. baumannii infection.

InvL, an Invasin-Like Adhesin, Is a Type II Secretion System Substrate Required for Acinetobacter baumannii Uropathogenesis.

Acinetobacter baumannii is an opportunistic pathogen of growing concern, as isolates are commonly multidrug resistant. While A. baumannii is most frequently associated with pulmonary infections, a significant proportion of clinical isolates come from urinary sources, highlighting its uropathogenic potential. The type II secretion system (T2SS) of commonly used model Acinetobacter strains is important for virulence in various animal models, but the potential role of the T2SS in urinary tract infection (UTI) remains unknown. Here, we used a catheter-associated UTI (CAUTI) model to demonstrate that a modern urinary isolate, UPAB1, requires the T2SS for full virulence. A proteomic screen to identify putative UPAB1 T2SS effectors revealed an uncharacterized lipoprotein with structural similarity to the intimin-invasin family, which serve as type V secretion system (T5SS) adhesins required for the pathogenesis of several bacteria. This protein, designated InvL, lacked the ß-barrel domain associated with T5SSs but was confirmed to require the T2SS for both surface localization and secretion. This makes InvL the first identified T2SS effector belonging to the intimin-invasin family. InvL was confirmed to be an adhesin, as the protein bound to extracellular matrix components and mediated adhesion to urinary tract cell lines in vitro. Additionally, the invL mutant was attenuated in the CAUTI model, indicating a role in Acinetobacter uropathogenesis. Finally, bioinformatic analyses revealed that InvL is present in nearly all clinical isolates belonging to international clone 2, a lineage of significant clinical importance. In all, we conclude that the T2SS substrate InvL is an adhesin required for A. baumannii uropathogenesis. IMPORTANCE While pathogenic Acinetobacter can cause various infections, we recently found that 20% of clinical isolates come from urinary sources. Despite the clinical relevance of Acinetobacter as a uropathogen, few virulence factors involved in urinary tract colonization have been defined. Here, we identify a novel type II secretion system effector, InvL, which is required for full uropathogenesis by a modern urinary isolate. Although InvL has predicted structural similarity to the intimin-invasin family of autotransporter adhesins, InvL is predicted to be anchored to the membrane as a lipoprotein. Similar to other invasin homologs, however, we demonstrate that InvL is a bona fide adhesin capable of binding extracellular matrix components and mediating adhesion to urinary tract cell lines. In all, this work establishes InvL as an adhesin important for Acinetobacter's urinary tract virulence and represents the first report of a type II secretion system effector belonging to the intimin-invasin family.

Interaction of Bartonella henselae with Fibronectin Represents the Molecular Basis for Adhesion to Host Cells.

Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host. IMPORTANCE Deciphering the mechanisms of bacterial host cell adhesion is a clue for preventing infections. We describe the underestimated role that the extracellular matrix protein fibronectin plays in the adhesion of human-pathogenic Bartonella henselae to host cells. Fibronectin-binding is mediated by a trimeric autotransporter adhesin (TAA) also present in many other human-pathogenic Gram-negative bacteria. We demonstrate that both TAA and host-fibronectin contribute significantly to bacterial adhesion, and we present the exact sequence of interacting amino acids from both proteins. Our work shows the domain-specific pattern of interaction between the TAA and fibronectin to adhere to host cells and opens the perspective to fight bacterial infections by inhibiting bacterial adhesion which represents generally the first step in infections.

Characterization of the Phase-Variable Autotransporter Lav Reveals a Role in Host Cell Adherence and Biofilm Formation in Nontypeable Haemophilus influenzae.

Lav is an autotransporter protein found in pathogenic Haemophilus and Neisseria species. Lav in nontypeable Haemophilus influenzae (NTHi) is phase-variable: the gene reversibly switches ON-OFF via changes in length of a locus-located GCAA(n) simple DNA sequence repeat tract. The expression status of lav was examined in carriage and invasive collections of NTHi, where it was predominantly not expressed (OFF). Phenotypic study showed lav expression (ON) results in increased adherence to human lung cells and denser biofilm formation. A survey of Haemophilus species genome sequences showed lav is present in ∼60% of NTHi strains, but lav is not present in most typeable H. influenzae strains. Sequence analysis revealed a total of five distinct variants of the Lav passenger domain present in Haemophilus spp., with these five variants showing a distinct lineage distribution. Determining the role of Lav in NTHi will help understand the role of this protein during distinct pathologies.