A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central “bridging organisms” in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive “early oral colonizers”. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation.
Fusobacterium nucleatum; RadD. Arginine; Adhesin; Biofilm; Co-aggregation
The periodontal pathogen Fusobacterium nucleatum induces apoptosis in lymphocytes. We previously identified the autotransporter protein Fap2 in F. nucleatum strain PK1594 that induced apoptosis in lymphocytes when expressed in Escherichia coli. In this study, we identified protein homologs of Fap2 in the transformable F. nucleatum strain ATCC 23726, to determine their role in the induction of apoptosis in lymphocytes. We used a new gene-inactivation vector conferring thiamphenicol resistance (pHS31) to construct a mutant deficient in one of the homologs, aim1. Transcriptional analyses demonstrated disruption of aim1 expression, and phenotypic analyses revealed a 41% decrease in the ability of the mutant to induce apoptosis in Jurkat cells, as compared with the parental strain. These studies demonstrate, in the native host cell background, the contribution of aim1 to F. nucleatum induction of apoptosis and, to the best of our knowledge, represent the first report of a genetically defined and phenotypically characterized mutation in F. nucleatum.
Fusobacterium nucleatum; apoptosis; autotransporter; aim1; mutant
Many bacterial components selectively activate immune and nonhematopoietic target cells via Toll-like receptor (TLR) signaling; modulation of such host responses defines the immune adjuvant properties of these bacterial products. For example, the outer membrane protein porins from Neisseria, Salmonella, and Shigella are known TLR2 agonists with established systemic and mucosal immune adjuvanticity. Early work indicated that the FomA porin from Fusobacterium nucleatum has immune adjuvant activity in mice. Using a purified recombinant FomA, we have verified its immune stimulatory properties and have defined a role for TLR2 signaling in its in vitro and in vivo activity. FomA induces interleukin 8 (IL-8) secretion and NF-κB-dependent luciferase activity in HEK cells expressing TLR2, IL-6 secretion, and cell surface upregulation of CD86 and major histocompatibility complex (MHC) II in primary B cells from wild-type mice, but it fails to activate cells from TLR2 knockout mice. Accordingly, the immune adjuvant activity of FomA is also TLR2 dependent. In a mouse model of immunization with ovalbumin (OVA), FomA induces enhanced production of OVA-specific IgM and IgG, including IgG1 and IgG2b antibodies, as well as enhanced secretion of IL-10 and IL-6, consistent with a Th2-type adjuvant effect. We also observe a moderate production of anti-FomA antibodies, suggesting that FomA is also immunogenic, a quality that is also TLR2 dependent. Therefore, modulation of host immune responses by FomA may be effective for targeting general host immunity not only to pathogens (as a novel TLR2 adjuvant) but also to F. nucleatum itself (as an antigen), expanding its use as a self-adjuvanted antigen in an immunization strategy against polymicrobial infections, including those by F. nucleatum.
The etiology of inflammatory bowel disease is not completely known, but it is influenced by the presence of normal gut microflora as well as yet-unrecognized pathogens. The anaerobic, Gram-negative bacterial species Fusobacterium nucleatum is a common resident of the human mouth and gut and varies in its pathogenic potential. In this study, we demonstrate that highly invasive F. nucleatum isolates derived from the inflamed guts of Crohn's disease patients evoked significantly greater MUC2 and tumor necrosis factor alpha (TNF-α) gene expression than minimally invasive strains isolated from the noninflamed gut in human colonic epithelial cells and in a rat ligated colonic loop model of infection. Only live F. nucleatum induced mucin secretion and TNF-α expression in direct contact with and/or during invasion of colonic cells. In rat colons, mucin secretion was augmented in response to a highly invasive F. nucleatum isolate but was unaffected by treatment with a minimally invasive strain. Taken together, these studies reveal that F. nucleatum may represent a challenging pathogen in the etiology of gut inflammatory diseases and highlight the importance of different pathotypes of candidate bacterial species in disease pathogenesis.
Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.
The mechanical therapy with multiple doses of antibiotics is one of modalities for treatment of periodontal diseases. However, treatments using multiple doses of antibiotics carry risks of generating resistant strains and misbalancing the resident body flora. We present an approach via immunization targeting an outer membrane protein FomA of Fusobacterium nucleatum, a central bridging organism in the architecture of oral biofilms. Neutralization of FomA considerably abrogated the enhancement of bacterial co-aggregation, biofilms and production of volatile sulfur compounds mediated by an interspecies interaction of F. nucleatum with Porphyromonas gingivalis (P. gingivalis). Vaccination targeting FomA also conferred a protective effect against co-infection-induced gum inflammation. Here, we advance a novel infectious mechanism by which F. nucleatum co-opts P. gingivalis to exacerbate gum infections. FomA is highlighted as a potential target for development of new therapeutics against periodontal infection and halitosis in humans.
Co-aggregation; Fusobacterium nucleatum; FomA; Porphyromonas gingivalis; Vaccine; Abscesses; Halitosis
Incubation of mammalian tumor cells with either soluble of insoluble fractions (10 to 100 micrograms/ml) of Fusobacterium nucleatum sensitizes them to the destructive activity of antibody-dependent cellular cytotoxicity (ADCC) effector cells in the presence of anti-F. nucleatum antisera. All three types of ADCC effector cells are capable of destroying F. nucleatum-sensitized target cells with varying degrees of effectiveness (lymphocytes much greater than monocytes greater than neutrophils). Hyperimmune rabbit anti-F. nucleatum antisera were active at a dilution as high as 1/100,000. Our studies indicated that F. nucleatum must be bound to the target cells since if either the effector cells are treated with F. nucleatum or F. nucleatum is directly to an ADCC reaction, there is no significant effect on cytotoxicity. The kinetics of F. nucleatum-targeted ADCC are identical to those of classical ADCC, suggesting a similar mechanism. The specificity of F. nucleatum-targeted ADCC was demonstrated by cold target inhibition studies and by showing that other antibacterial antisera were incapable of mediating the activity.
Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air-liquid interface in low calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. P. gingivalis invaded intracellularly and spread cell to cell. A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer. S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced IL-1β, TNF-α, IL-6 and IL-8 secretion; and F. nucleatum stimulated production of IL-1β and TNF-α. A. actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, while the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.
oral pathogens; oral commensals; virulence; periodontal disease
The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.
Gram-negative bacterial proteins which are exported from the cytosol to the external environment by the type V secretion system are also known as autotransporters. Once translocated to the periplasmic compartment by the sec-dependent general secretory pathway, their C-terminal domain forms a pore through which the N-terminal domain travels to the outer membrane without the need of other accessory proteins. MisL (protein of membrane insertion and secretion) is a protein of unknown function located in the pathogenicity island SPI-3 of Salmonella enterica and classified as an autotransporter due to its high homology to Escherichia coli AIDA-I. In the present work, the MisL C-terminal translocator domain was used to display the immunodominant B-cell epitope of the circumsporozoite protein (CSP) from Plasmodium falciparum on the surface of Salmonella enterica serovar Typhimurium (serovar Typhimurium SL3261) and serovar Typhi (serovar Typhi CVD 908). The MisL β domain was predicted by alignment with AIDA-I, amplified from serovar Typhimurium SL3261, cloned in a plasmid fused to four repeats of the tetrapeptide NANP behind the Escherichia coli heat-labile enterotoxin B subunit signal peptide to ensure periplasmic traffic, and expressed under the control of the anaerobically inducible nirB promoter. The fusion protein was translocated to the outer membrane of both bacterial strains, although the foreign epitope was displayed more efficiently in serovar Typhimurium SL3261, which elicited a better specific antibody response in BALB/c mice. More importantly, antibodies were able to recognize the native CSP in P. falciparum sporozoites. These results confirm that MisL is indeed an autotransporter and that it can be used to express foreign immunogenic epitopes on the surface of gram-negative bacteria.
Intimin is a bacterial adhesin located on the surface of enteropathogenic Escherichia coli and other related bacteria that is believed to self-translocate across the outer membrane (OM), and therefore it has been regarded as a member of the type V secretion system (T5SS), which includes classical autotransporters (ATs). However, intimin has few structural similarities to classical ATs and an opposite topology with an OM-embedded N region and a secreted C region. Since the actual secretion mechanism of intimin is unknown, we investigated intimin biogenesis by analyzing its requirement of periplasmic chaperones (DsbA, SurA, Skp, and DegP) and of OM protein BamA (YaeT/Omp85) for folding, OM insertion, and translocation. Using full-length and truncated intimin polypeptides, we demonstrate that DsbA catalyzes the formation of a disulfide bond in the D3 lectin-like domain of intimin in the periplasm, indicating that this secreted C-terminal domain is at least partially folded prior to its translocation across the OM. We also show that SurA chaperone plays the major role for periplasmic transport and folding of the N region of intimin, whereas the parallel pathway made by Skp and DegP chaperones plays a secondary role in this process. Further, we demonstrate that BamA is essential for the insertion of the N region of intimin in the OM and that the protease activity of DegP participates in the degradation of misfolded intimin. The significance of these findings for a BamA-dependent secretion mechanism of intimin is discussed in the context of T5SSs.
Cells of the innate immune system regulate immune responses through the production of antimicrobial peptides, chemokines, and cytokines, including human beta-defensins (hBDs) and CCL20. In this study, we examined the kinetics of primary human oral epithelial cell (HOEC) production of CCL20 and hBDs in response to Fusobacterium nucleatum, a commensal bacterium of the oral cavity, which we previously showed promotes HOEC induction of hBDs. HOECs secrete maximal levels of CCL20 at 6 h, following stimulation by F. nucleatum cell wall (FnCW). The kinetics of CCL20 release is distinct from that of hBD-2 and -3, which peaks after 24 h and 48 h of FnCW stimulation, respectively. FnCW-induced release of CCL20 by HOECs requires both transcriptional and translational activation. Release of CCL20 by HOECs is inhibited by brefeldin A, suggesting that it is secreted through a vesicle transport pathway. Other epithelium-derived agents that FnCW induces, such as hBD-2, hBD-3, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), are also able to release CCL20. By focusing on mitogen-activated protein kinases, we show that both extracellular signal-regulated kinase 1/2 and p38, but not JNK, are required for hBD-, TNF-α-, and IL-1β-induced secretion of CCL20 by HOECs. The ability of FnCW and its induced hBDs to produce proinflammatory cytokines and CCL20 suggests the broad role of F. nucleatum and human antimicrobial peptides in primary immune responses elicited by oral epithelium.
Septic shock due to Salmonella and other gram-negative enteric pathogens is a leading cause of death worldwide. The role of lipopolysaccharide in sepsis is well studied; however, the contribution of other bacterial outer membrane components, such as Braun (murein) lipoprotein (Lpp), is not well defined. The genome of Salmonella enterica serovar Typhimurium harbors two copies of the lipoprotein (lpp) gene. We constructed a serovar Typhimurium strain with deletions in both copies of the lpp gene (lpp1 and lpp2) by marker exchange mutagenesis. The integrity of the cell membrane and the secretion of the effector proteins through the type III secretion system were not affected in the lpp double-knockout mutant. Subsequently, the virulence potential of this mutant was examined in a cell culture system using T84 intestinal epithelial and RAW264.7 macrophage cell lines and a mouse model of salmonellosis. The lpp double-knockout mutant was defective in invading and inducing cytotoxic effects in T84 and RAW264.7 cells, although binding of the mutant to the host cell was not affected when compared to the wild-type (WT) serovar Typhimurium. The motility of the mutant was impaired, despite the finding that the number of flagella was similar in the lpp double knockout mutant and the WT serovar Typhimurium. Deletion in the lpp genes did not affect the intracellular survival and replication of Salmonella in macrophages and T84 cells. Induction of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-8 (IL-8) was significantly reduced in macrophages and T84 cells infected with the lpp double-knockout mutant. The levels of IL-8 remained unaffected in T84 cells when infected with either live or heat-killed WT and lpp mutant, indicating that invasion was not required for IL-8 production and that Toll-like receptor 2 signaling might be affected in the Lpp double-knockout mutant. These effects of the Lpp protein could be restored by complementation of the isogenic mutant. The lpp double-knockout mutant was avirulent in mice, and animals infected with this mutant were protected from a lethal challenge dose of WT serovar Typhimurium. The severe combined immunodeficient mice, on the other hand, were susceptible to infection by the lpp double-knockout mutant. The serovar Typhimurium mutants from which only one of the lpp (lpp1 or lpp2) genes was deleted were also avirulent in mice. Taken together, our data indicated that Lpp specifically contributed to the virulence of the organism.
Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function.
IcsA is an outer membrane protein in the autotransporter family that is required for Shigella flexneri pathogenesis. Following its secretion through the Sec translocon, IcsA is incorporated into the outer membrane in a process that depends on YaeT, a component of an outer membrane β-barrel insertion machinery. We investigated the role of the periplasmic chaperone Skp in IcsA maturation. Skp is required for the presentation of the mature amino terminus (alpha-domain) of IcsA on the bacterial surface and contributes to cell-to-cell spread of S. flexneri in cell culture. A mutation in skp does not prevent the insertion of the β-barrel into the outer membrane, suggesting that the primary role of Skp is the folding of the IcsA alpha-domain. In addition, the requirement for skp can be partially bypassed by disrupting icsP, an ortholog of Escherichia coli ompT, which encodes the protease that processes IcsA between the mature amino terminus and the β-barrel outer membrane anchor. These findings are consistent with a model in which Skp plays a critical role in the chaperoning of the alpha-domain of IcsA during transit through the periplasm.
Bacterial virulence mechanisms are attractive targets for antibiotic development, because they are required for the pathogenesis of numerous global infectious disease agents. The bacterial secretion systems used to assemble the surface structures that promote adherence and deliver protein virulence effectors to host cells could comprise one such therapeutic target. In this study, we developed and performed a high-throughput screen (HTS) of small molecule libraries and identified a small molecule, a 2-imino-5-arylidene thiazolidinone that blocked secretion and virulence functions of a wide array of animal and plant Gram-negative bacterial pathogens. This compound inhibited type III secretion-dependent functions, with the exception of flagellar motility, and Type II secretion-dependent functions, suggesting that the target of the compound could be an outer membrane component conserved between these two secretion systems. This work provides a proof of concept that compounds with a broad spectrum of activity against Gram-negative bacterial secretion systems could be developed to prevent and treat bacterial diseases.
Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.
The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is required for systemic infections and intracellular accumulation of Salmonella enterica. This system is induced by intracellular Salmonella and subsequently transfers effector proteins into the host cell. Growth conditions either inducing expression of the type III secretion system or the secretion of substrate proteins were defined. Here we report the identification of a set of substrate proteins consisting of SseB, SseC, and SseD that are secreted by the SPI2 system in vitro. Secretion was observed if bacterial cells were exposed to acidic pH after growth in minimal medium with limitation of Mg2+ or phosphate. SseB, -C, and -D were isolated in a fraction detached from the bacterial cell surface by mechanical shearing, indicating that these proteins are predominantly assembled into complexes on the bacterial cell surface. The three proteins were required for the translocation of SPI2 effector proteins SspH1 and SspH2 into infected host cells. Thus, SseB, SseC, and SseD function as the translocon for effector proteins by intracellular Salmonella.
Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters.
Bacterial adaptation to external stresses and toxic compounds is a key step in the emergence of multidrug-resistant strains that are a serious threat to human health. Although some of the proteins and regulators involved in antibiotic resistance mechanisms have been described, no information is available to date concerning the early bacterial response to external stresses. Here we report that the expression of ompX, encoding an outer membrane protein, is increased during early exposure to drugs or environmental stresses. At the same time, the level of ompF porin expression is noticeably affected. Because of the role of these proteins in membrane permeability, these data suggest that OmpF and OmpX are involved in the control of the penetration of antibiotics such as β-lactams and fluoroquinolones through the enterobacterial outer membrane. Consequently, the early control of ompX and ompF induced by external stresses may represent a preliminary response to antibiotics, thus triggering the initial bacterial line of defense against antibiotherapy.
Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved. While OmpA was required for bacterial binding to polarized Caco-2 cells, it was not needed for the induction of cytokine production by Caco-2 cells or for human DC migration across polarized cells.
The dental plaque is comprised of numerous bacterial species which may or may not be pathogenic. Human gingival epithelial cells (HGECs) respond to perturbation by various bacteria of the dental plaque by production of different levels of inflammatory cytokines which is a putative reflection of their virulence. The aim of the current study was to determine responses in terms of IL-1β, IL-6, IL-8 and IL-10 secretion induced by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus gordonii in order to gauge their virulence potential.
Materials and Methods
HGECs were challenged with the four bacterial species, live or heat-killed, at various MOIs (multiplicity of infection) and the elicited IL-1β, IL-6, IL-8 and IL-10 responses were assayed by ELISA.
Primary HGECs challenged with live P. gingivalis produced high levels of IL-1β, while challenge with live A. actinomycetemcomitans gave high levels of IL-8. The opportunistic pathogen F. nucleatum induces the highest levels of pro-inflammatory cytokines, while the commensal S. gordonii is the least stimulatory.
We conclude that various dental plaque biofilm bacteria induce different cytokine response profiles in primary human gingival epithelial cells that may reflect their individual virulence or commensal status.
P. gingivalis; A. actinomycetemcomitans; F. nucleatum; S.gordonii; epithelial cells; cytokines
Type III secretion is used by many gram-negative bacterial pathogens to directly deliver protein toxins (effectors) into targeted host cells. In all cases, secretion of effectors is triggered by host cell contact, although the mechanism is unclear. In Pseudomonas aeruginosa, expression of all type III secretion-related genes is up-regulated when secretion is triggered. We were able to visualize this process using a green fluorescent protein reporter system and to use it to monitor the ability of bacteria to trigger effector secretion on cell contact. Surprisingly, the action of one of the major type III secreted effectors, ExoS, prevented triggering of type III secretion by bacteria that subsequently attached to cells, suggesting that triggering of secretion is feedback regulated. Evidence is presented that translocation (secretion of effectors across the host cell plasma membrane) of ExoS is indeed self-regulated and that this inhibition of translocation can be achieved by either of its two enzymatic activities. The translocator proteins PopB, PopD, and PcrV are secreted via the type III secretion system and are required for pore formation and translocation of effectors across the host cell plasma membrane. Here we present data that secretion of translocators is in fact not controlled by calcium, implying that triggering of effector secretion on cell contact represents a switch in secretion specificity, rather than a triggering of secretion per se. The requirement for a host cell cofactor to control effector secretion may help explain the recently observed phenomenon of target cell specificity in both the Yersinia and P. aeruginosa type III secretion systems.
Autotransporters of gram-negative bacteria are single-peptide secretion systems that consist of a functional N-terminal α-domain (“passenger”) fused to a C-terminal β-domain (“translocator”). How passenger proteins are translocated through the outer membrane has not been resolved, and at present essentially three different models are discussed. In the widely accepted “hairpin model” the passenger proteins are translocated through a channel formed by the β-barrel of the translocator that is integrated in the outer membrane. This model has been challenged by a recent proposal for a general autotransporter model suggesting that there is a hexameric translocation pore that is generated by the oligomerization of six β-domains. A third model suggests that conserved Omp85 participates in autotransporter integration and passenger protein translocation. To examine these models, in this study we investigated the presence of putative oligomeric structures of the translocator of the autotransporter adhesin involved in diffuse adherence (AIDA) in vivo by cross-linking techniques. Furthermore, the capacity of isolated AIDA fusion proteins to form oligomers was studied in vitro by several complementary analytical techniques, such as analytical gel filtration, electron microscopy, immunogold labeling, and cross-linking of recombinant autotransporter proteins in which different passenger proteins were fused to the AIDA translocator. Our results show that the AIDA translocator is mostly present as a monomer. Only a fraction of the AIDA autotransporter was found to form dimers on the bacterial surface and in solution. Higher-order structures, such as hexamers, were not detected either in vivo or in vitro and can therefore be excluded as functional moieties for the AIDA autotransporter.
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are attaching and effacing (A/E) bacterial pathogens that cause severe diarrheal disease worldwide. To cause disease, A/E pathogens require a type III secretion system, which facilitates transport of bacterial effector proteins directly into infected host cells. One of these effector proteins translocated by the type III secretion system, EspZ, is essential for A/E pathogen infection and functions to prevent rapid death of EPEC-infected cells. We further investigated the mechanism of EspZ-mediated protection of infected host cells and found that a severe decrease in host mitochondrial membrane potential (Δψm) occurs concurrently with host cell lysis during infection with EPEC lacking EspZ (ΔespZ). It was also demonstrated that EspZ localizes to host cell mitochondria and interacts with the translocase of inner mitochondrial membrane 17b (TIM17b). In addition, host cell cytotoxicity was exacerbated in the absence of TIM17b during wild-type (WT) EPEC infection. The findings of this study together provide the first evidence that EspZ localizes to host mitochondria and that TIM17b contributes to protection against rapid cell death during EPEC infection.