Bartonella type IV secretion effector BepC induces stress fiber formation through activation of GEF-H1.

Bartonella T4SS effector BepC was reported to mediate internalization of big Bartonella aggregates into host cells by modulating F-actin polymerization. After that, BepC was indicated to induce host cell fragmentation, an interesting cell phenotype that is characterized by failure of rear-end retraction during cell migration, and subsequent dragging and fragmentation of cells. Here, we found that expression of BepC resulted in significant stress fiber formation and contractile cell morphology, which depended on combination of the N-terminus FIC (filamentation induced by c-AMP) domain and C-terminus BID (Bartonella intracellular delivery) domain of BepC. The FIC domain played a key role in BepC-induced stress fiber formation and cell fragmentation because deletion of FIC signature motif or mutation of two conserved amino acid residues abolished BepC-induced cell fragmentation. Immunoprecipitation confirmed the interaction of BepC with GEF-H1 (a microtubule-associated RhoA guanosine exchange factor), and siRNA-mediated depletion of GEF-H1 prevented BepC-induced stress fiber formation. Interaction with BepC caused the dissociation of GEF-H1 from microtubules and activation of RhoA to induce formation of stress fibers. The ROCK (Rho-associated protein kinase) inhibitor Y27632 completely blocked BepC effects on stress fiber formation and cell contractility. Moreover, stress fiber formation by BepC increased the stability of focal adhesions, which consequently impeded rear-edge detachment. Overall, our study revealed that BepC-induced stress fiber formation was achieved through the GEF-H1/RhoA/ROCK pathway.

Bartonella quintana type IV secretion effector BepE-induced selective autophagy by conjugation with K63 polyubiquitin chain.

Bartonella effector proteins (named Beps) are substrates of VirB type IV secretion system for translocation into host cells evolved in Bartonella spp. Among these, BepE has been shown to protect cells from fragmentation effects triggered by other Beps and to promote in vivo dissemination of bacteria from the dermal site of inoculation to the bloodstream. Bacterial pathogens secreted effectors to modulate the interplay with host autophagy, either to combat autophagy to escape its bactericidal effect or to exploit autophagy to benefit intracellular replication. Here, we reported a distinct phenotype that selective autophagy in host cells is activated as a countermeasure, to attack BepE via conjugation with K63 polyubiquitin chain on BepE. We found that ectopic expression of Bartonella quintana BepE specifically induced punctate structures that colocalised with an autophagy marker (LC3-II) in host cells, in addition to filopodia and membrane ruffle formation. Two tandemly arranged Bartonella Intracellular Delivery (BID) domains in the BepE C-terminus, where ubiquitination of sister pairs of lysine residues was confirmed, were essential to activate host cell autophagy. Multiple polyubiquitin chain linkages of K27, K29, K33, and K63 were found to be conjugated at sites of K222 and K365 on BepE, of which K63 polyubiquitination on BepE K365 determined the selective autophagy (p62/SQSTM1 positive autophagy) independent of the PI3K pathway. Colocalisation of BepE with LAMP1 confirmed the maturation of BepE-induced autophagosomes in which BepE were targeted for degradation. Moreover, host cells employed selective autophagy to counter-attack BepE to rescue cells from BepE-induced endocytosis deficiency.

Dynamics of Co-Infection with Bartonella henselae Genotypes I and II in Naturally Infected Cats: Implications for Feline Vaccine Development.

Bartonella henselae is an emerging bacterial pathogen causing cat-scratch disease and potentially fatal bacillary angiomatosis in humans. Bacteremic cats constitute a large reservoir for human infection. Although feline vaccination is a potential strategy to prevent human infection, selection of appropriate B. henselae strains is critical for successful vaccine development. Two distinct genotypes of B. henselae (type I, type II) have been identified and are known to co-infect the feline host, but very little is known about the interaction of these two genotypes during co-infection in vivo. To study the in vivo dynamics of type I and type II co-infection, we evaluated three kittens that were naturally flea-infected with both B. henselae type I and type II. Fifty individual bloodstream isolates from each of the cats over multiple time points were molecularly typed (by 16S rRNA gene sequencing), to determine the prevalence of the two genotypes over 2 years of persistent infection. We found that both B. henselae genotypes were transmitted simultaneously to each cat via natural flea infestation, resulting in mixed infection with both genotypes. Although the initial infection was predominately type I, after the first 2 months, the isolated genotype shifted to exclusively type II, which then persisted with a relapsing pattern. Understanding the parameters of protection against both genotypes of B. henselae, and the competitive dynamics in vivo between the two genotypes, will be critical in the development of a successful feline vaccine that can ultimately prevent B. henselae transmission to human contacts.

Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonellakoehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats.

Domestic cats are the natural reservoir of Bartonella henselae, B. clarridgeiae and B. koehlerae. To determine the role of wild felids in the epidemiology of Bartonella infections, blood was collected from 14 free-ranging California mountain lions (Puma concolor) and 19 bobcats (Lynx rufus). Bartonella spp. were isolated from four (29%) mountain lions and seven (37%) bobcats. These isolates were characterized using growth characteristics, biochemical reactions, molecular techniques, including PCR-RFLP of selected genes or interspacer region, pulsed-field gel electrophoresis (PFGE), partial sequencing of several genes, and DNA-DNA hybridization. Two isolates were identical to B. henselae genotype II. All other isolates were distinguished from B. henselae and B. koehlerae by PCR-RFLP of the gltA gene using endonucleases HhaI, TaqI and AciI, with the latter two discriminating between the mountain lion and the bobcat isolates. These two novel isolates displayed specific PFGE profiles distinct from B. henselae, B. koehlerae and B. clarridgeiae. Sequences of amplified gene fragments from the three mountain lion and six bobcat isolates were closely related to, but distinct from, B. henselae and B. koehlerae. Finally, DNA-DNA hybridization studies demonstrated that the mountain lion and bobcat strains are most closely related to B. koehlerae. We propose naming the mountain lion isolates B. koehlerae subsp. boulouisii subsp. nov. (type strain: L-42-94), and the bobcat isolates B. koehlerae subsp. bothieri subsp. nov. (type strain: L-17-96), and to emend B. koehlerae as B. koehlerae subsp. koehlerae. The mode of transmission and the zoonotic potential of these new Bartonella subspecies remain to be determined.

Bartonella quintana Aortitis in a Man with AIDS, Diagnosed by Needle Biopsy and 16S rRNA Gene Amplification.

A man with newly diagnosed AIDS presented with months of back pain and fever. Computed tomography (CT) results demonstrated aortitis with periaortic tissue thickening. DNA amplification of biopsy tissue revealed Bartonella quintana, and Bartonella serologies were subsequently noted to be positive. The patient improved with prolonged doxycycline and rifabutin treatment. This case illustrates how molecular techniques are increasingly important in diagnosing Bartonella infections.

Experimental infection of cats with Afipia felis and various Bartonella species or subspecies.

Based upon prior studies, domestic cats have been shown to be the natural reservoir for Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. However, other Bartonella species, such as Bartonella vinsonii subsp. berkhoffii, Bartonella quintana or Bartonella bovis (ex weissii) have been either isolated from or Bartonella DNA sequences PCR amplified and sequenced. In the late 1980s, before B. henselae was confirmed as the etiological agent of cat scratch disease, Afipia felis had been proposed as the causative agent. In order to determine the feline susceptibility to A. felis, B. vinsonii subsp. berkhoffii, Bartonella rochalimae, B. quintana or B. bovis, we sought to detect the presence of bacteremia and seroconversion in experimentally-inoculated cats. Most of the cats seroconverted, but only the cats inoculated with B. rochalimae became bacteremic, indicating that cats are not natural hosts of A. felis or the other Bartonella species or subspecies tested in this study.

Bartonella quintana deploys host and vector temperature-specific transcriptomes.

The bacterial pathogen Bartonella quintana is passed between humans by body lice. B. quintana has adapted to both the human host and body louse vector niches, producing persistent infection with high titer bacterial loads in both the host (up to 10(5) colony-forming units [CFU]/ml) and vector (more than 10(8) CFU/ml). Using a novel custom microarray platform, we analyzed bacterial transcription at temperatures corresponding to the host (37°C) and vector (28°C), to probe for temperature-specific and growth phase-specific transcriptomes. We observed that transcription of 7% (93 genes) of the B. quintana genome is modified in response to change in growth phase, and that 5% (68 genes) of the genome is temperature-responsive. Among these transcriptional changes in response to temperature shift and growth phase was the induction of known B. quintana virulence genes and several previously unannotated genes. Hemin binding proteins, secretion systems, response regulators, and genes for invasion and cell attachment were prominent among the differentially-regulated B. quintana genes. This study represents the first analysis of global transcriptional responses by B. quintana. In addition, the in vivo experiments provide novel insight into the B. quintana transcriptional program within the body louse environment. These data and approaches will facilitate study of the adaptation mechanisms employed by Bartonella during the transition between human host and arthropod vector.

The Bartonella quintana extracytoplasmic function sigma factor RpoE has a role in bacterial adaptation to the arthropod vector environment.

Bartonella quintana is a vector-borne bacterial pathogen that causes fatal disease in humans. During the infectious cycle, B. quintana transitions from the hemin-restricted human bloodstream to the hemin-rich body louse vector. Because extracytoplasmic function (ECF) sigma factors often regulate adaptation to environmental changes, we hypothesized that a previously unstudied B. quintana ECF sigma factor, RpoE, is involved in the transition from the human host to the body louse vector. The genomic context of B. quintana rpoE identified it as a member of the ECF15 family of sigma factors found only in alphaproteobacteria. ECF15 sigma factors are believed to be the master regulators of the general stress response in alphaproteobacteria. In this study, we examined the B. quintana RpoE response to two stressors that are encountered in the body louse vector environment, a decreased temperature and an increased hemin concentration. We determined that the expression of rpoE is significantly upregulated at the body louse (28°C) versus the human host (37°C) temperature. rpoE expression also was upregulated when B. quintana was exposed to high hemin concentrations. In vitro and in vivo analyses demonstrated that RpoE function is regulated by a mechanism involving the anti-sigma factor NepR and the response regulator PhyR. The ΔrpoE ΔnepR mutant strain of B. quintana established that RpoE-mediated transcription is important in mediating the tolerance of B. quintana to high hemin concentrations. We present the first analysis of an ECF15 sigma factor in a vector-borne human pathogen and conclude that RpoE has a role in the adaptation of B. quintana to the hemin-rich arthropod vector environment.

Rasamsonia argillacea pulmonary and aortic graft infection in an immune-competent patient.

Rasamsonia argillacea (formerly known as Geosmithia argillacea) is a fungus recently recognized as a pathogen of immunocompromised patients. Here we report the first case of Rasamsonia infection in an immunocompetent host, presenting as a pulmonary and aortic graft infection. Its morphological similarity to nonpathogenic Penicillium species delayed the diagnosis and initiation of appropriate treatment.

Hemin binding protein C is found in outer membrane vesicles and protects Bartonella henselae against toxic concentrations of hemin.

Bartonella species are gram-negative, emerging bacterial pathogens found in two distinct environments. In the gut of the obligately hematophagous arthropod vector, bartonellae are exposed to concentrations of heme that are toxic to other bacteria. In the bloodstream of the mammalian host, access to heme and iron is severely restricted. Bartonellae have unusually high requirements for heme, which is their only utilizable source of iron. Although heme is essential for Bartonella survival, little is known about genes involved in heme acquisition and detoxification. We developed a strategy for high-efficiency transposon mutagenesis to screen for genes in B. henselae heme binding and uptake pathways. We identified a B. henselae transposon mutant that constitutively expresses the hemin binding protein C (hbpC) gene. In the wild-type strain, transcription of B. henselae hbpC was upregulated at arthropod temperature (28°C), compared to mammalian temperature (37°C). In the mutant strain, temperature-dependent regulation was absent. We demonstrated that HbpC binds hemin and localizes to the B. henselae outer membrane and outer membrane vesicles. Overexpression of hbpC in B. henselae increased resistance to heme toxicity, implicating HbpC in protection of B. henselae from the toxic levels of heme present in the gut of the arthropod vector. Experimental inoculation of cats with B. henselae strains demonstrated that both constitutive expression and deletion of hbpC affect the ability of B. henselae to infect the cat host. Modulation of hbpC expression appears to be a strategy employed by B. henselae to survive in the arthropod vector and the mammalian host.

Identification of the feline humoral immune response to Bartonella henselae infection by protein microarray.

BACKGROUND: Bartonella henselae is the zoonotic agent of cat scratch disease and causes potentially fatal infections in immunocompromised patients. Understanding the complex interactions between the host's immune system and bacterial pathogens is central to the field of infectious diseases and to the development of effective diagnostics and vaccines. METHODOLOGY: We report the development of a microarray comprised of proteins expressed from 96% (1433/1493) of the predicted ORFs encoded by the genome of the zoonotic pathogen Bartonella henselae. The array was probed with a collection of 62 uninfected, 62 infected, and 8 "specific-pathogen free" naïve cat sera, to profile the antibody repertoire elicited during natural Bartonella henselae infection. CONCLUSIONS: We found that 7.3% of the B. henselae proteins on the microarray were seroreactive and that seroreactivity was not evenly distributed between predicted protein function or subcellular localization. Membrane proteins were significantly most likely to be seroreactive, although only 23% of the membrane proteins were reactive. Conversely, we found that proteins involved in amino acid transport and metabolism were significantly underrepresented and did not contain any seroreactive antigens. Of all seroreactive antigens, 52 were differentially reactive with sera from infected cats, and 53 were equally reactive with sera from infected and uninfected cats. Thirteen of the seroreactive antigens were found to be differentially seroreactive between B. henselae type I and type II. Based on these results, we developed a classifier algorithm that was capable of accurately discerning 93% of the infected animals using the microarray platform. The seroreactivity and diagnostic potential of these antigens was then validated on an immunostrip platform, which correctly identified 98% of the infected cats. Our protein microarray platform provides a high-throughput, comprehensive analysis of the feline humoral immune response to natural infection with the alpha-proteobacterium B. henselae at an antigen-specific, sera-specific, and genome-wide level. Furthermore, these results provide novel insight and utility in diagnostics, vaccine development, and understanding of host-pathogen interaction.

Molecular epidemiology of feline and human Bartonella henselae isolates.

Multiple locus variable number tandem repeat analysis was performed on 178 Bartonella henselae isolates from 9 countries; 99 profiles were distributed into 2 groups. Human isolates/strains were placed into the second group. Genotype I and II isolates shared no common profile. All genotype I isolates clustered within group B. The evolutive implications are discussed.

Ecological fitness and strategies of adaptation of Bartonella species to their hosts and vectors.

Bartonella spp. are facultative intracellular bacteria that cause characteristic hostrestricted hemotropic infections in mammals and are typically transmitted by blood-sucking arthropods. In the mammalian reservoir, these bacteria initially infect a yet unrecognized primary niche, which seeds organisms into the blood stream leading to the establishment of a long-lasting intra-erythrocytic bacteremia as the hall-mark of infection. Bacterial type IV secretion systems, which are supra-molecular transporters ancestrally related to bacterial conjugation systems, represent crucial pathogenicity factors that have contributed to a radial expansion of the Bartonella lineage in nature by facilitating adaptation to unique mammalian hosts. On the molecular level, the type IV secretion system VirB/VirD4 is known to translocate a cocktail of different effector proteins into host cells, which subvert multiple cellular functions to the benefit of the infecting pathogen. Furthermore, bacterial adhesins mediate a critical, early step in the pathogenesis of the bartonellae by binding to extracellular matrix components of host cells, which leads to firm bacterial adhesion to the cell surface as a prerequisite for the efficient translocation of type IV secretion effector proteins. The best-studied adhesins in bartonellae are the orthologous trimeric autotransporter adhesins, BadA in Bartonella henselae and the Vomp family in Bartonella quintana. Genetic diversity and strain variability also appear to enhance the ability of bartonellae to invade not only specific reservoir hosts, but also accidental hosts, as shown for B. henselae. Bartonellae have been identified in many different blood-sucking arthropods, in which they are typically found to cause extracellular infections of the mid-gut epithelium. Adaptation to specific vectors and reservoirs seems to be a common strategy of bartonellae for transmission and host diversity. However, knowledge regarding arthropod specificity/restriction, the mode of transmission, and the bacterial factors involved in arthropod infection and transmission is still limited.

Dogs are more permissive than cats or guinea pigs to experimental infection with a human isolate of Bartonella rochalimae.

Bartonella rochalimae was first isolated from the blood of a human who traveled to Peru and was exposed to multiple insect bites. Foxes and dogs are likely natural reservoirs for this bacterium. We report the results of experimental inoculation of two dogs, five cats and six guinea pigs with the only human isolate of this new Bartonella species. Both dogs became bacteremic for 5-7 weeks, with a peak of 10(3)-10(4) colony forming units (CFU)/mL blood. Three cats had low bacteremia levels (< 200 CFU/mL) of 6-8 weeks' duration. One cat that remained seronegative had two bacterial colonies isolated at a single culture time point. A fifth cat never became bacteremic, but seroconverted. None of the guinea pigs became bacteremic, but five seroconverted. These results suggest that dogs could be a reservoir of this strain of B. rochalimae, in contrast to cats and guinea pigs.

Infective endocarditis in a dog and the phylogenetic relationship of the associated "Bartonella rochalimae" strain with isolates from dogs, gray foxes, and a human.

The first case of canine endocarditis caused by "Bartonella rochalimae" is reported. By PCR-restriction fragment length polymorphism, sequence, and phylogenetic analyses, Bartonella isolates from a dog with endocarditis, 22 gray foxes, and three dogs, described as B. clarridgeiae like, were confirmed to belong to the new species "B. rochalimae," suggesting canids as the natural reservoir.

A SacB mutagenesis strategy reveals that the Bartonella quintana variably expressed outer membrane proteins are required for bloodstream infection of the host.

Bartonella bacteria adhere to erythrocytes and persistently infect the mammalian bloodstream. We previously identified four highly conserved Bartonella quintana adhesin genes that undergo phase variation during prolonged bloodstream infection. The variably expressed outer membrane proteins (Vomp) encoded by these genes are members of the trimeric autotransporter adhesin family. Each B. quintana Vomp appears to contribute a different adhesion phenotype, likely mediated by the major variable region at the adhesive tip of each Vomp. Although studies document that the Vomp adhesins confer virulence phenotypes in vitro, little is known about in vivo virulence strategies of Bartonella. We sought to determine whether the B. quintana Vomp adhesins are necessary for infection in vivo by using a vomp null mutant. It first was necessary to develop a system to generate in-frame deletions of defined genes by allelic exchange in a wild-type Bartonella background, which had not been achieved previously. We utilized sacB negative selection to generate a targeted, in-frame, markerless deletion of the entire vomp locus in B. quintana. We also recently developed the first animal model for B. quintana infection, and using this model, we demonstrate here that the deletion of the entire vomp locus, but not the deletion of two vomp genes, results in a null mutant strain that is incapable of establishing bloodstream infection in vivo. The Vomp adhesins therefore represent critical virulence factors in vivo, warranting further study. Finally, our allelic exchange strategy provides an important advance in the genetic manipulation of all Bartonella species and, combined with the animal model that recapitulates human disease, will facilitate pathogenesis studies of B. quintana.

A survey of veterinarian involvement in zoonotic disease prevention practices.

OBJECTIVE: To determine the extent to which practicing veterinarians in King County, Washington, engaged in commonly recommended practices for the prevention of zoonotic diseases. DESIGN: Cross-sectional survey. Sample Population-Licensed veterinarians practicing clinical medicine in King County, Washington. PROCEDURES: A survey was sent between September and November 2006 to 454 licensed veterinarians practicing clinical medicine in King County. RESULTS: 370 valid responses were received. A high proportion (280/362 [77%]) of respondents agreed that it was very important for veterinarians to educate clients on zoonotic disease prevention, but only 43% (158/367) reported that they had initiated discussions about zoonotic diseases with clients on a daily basis, and only 57% (203/356) indicated that they had client educational materials on zoonotic diseases available in their practices. Thirty-one percent (112/360) of respondents indicated that there were no written infection-control guidelines for staff members in the practice, and 28% (105/371) reported having been infected with a zoonotic disease in practice. CONCLUSIONS AND CLINICAL RELEVANCE: Results illustrated that veterinarians recognize their important role in zoonotic disease prevention and suggested that veterinarians would welcome stronger partnerships with public health agencies and other health professionals in this endeavor. Methods to increase veterinarians' involvement in zoonotic disease prevention include discussing zoonotic diseases more frequently with clients, physicians, and public health agencies; encouraging higher risk individuals to discuss zoonotic diseases; having educational materials on zoonotic diseases available for clients; improving infection-control practices; and ensuring that continuing education courses on zoonotic diseases are regularly available.

Bacteremia, fever, and splenomegaly caused by a newly recognized bartonella species.

Bartonella species cause serious human infections globally, including bacillary angiomatosis, Oroya fever, trench fever, and endocarditis. We describe a patient who had fever and splenomegaly after traveling to Peru and also had bacteremia from an organism that resembled Bartonella bacilliformis, the causative agent of Oroya fever, which is endemic to Peru. However, genetic analyses revealed that this fastidious bacterium represented a previously uncultured and unnamed bartonella species, closely related to B. clarridgeiae and more distantly related to B. bacilliformis. We characterized this isolate, including its ability to cause fever and sustained bacteremia in a rhesus macaque. The route of infection and burden of human disease associated with this newly described pathogen are currently unknown.

Proteomic and immunoblot analyses of Bartonella quintana total membrane proteins identify antigens recognized by sera from infected patients.

Bartonella quintana is a fastidious, gram-negative, rod-shaped bacterium that causes prolonged bacteremia in immunocompetent humans and severe infections in immunocompromised individuals. We sought to define the outer membrane subproteome of B. quintana in order to obtain insight into the biology and pathogenesis of this emerging pathogen and to identify the predominant B. quintana antigens targeted by the human immune system during infection. We isolated the total membrane proteins of B. quintana and identified 60 proteins by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass fingerprinting. Using the newly constructed proteome map, we then utilized two-dimensional immunoblotting with sera from 21 B. quintana-infected patients to identify 24 consistently recognized, immunoreactive B. quintana antigens that have potential relevance for pathogenesis and diagnosis. Among the outer membrane proteins, the variably expressed outer membrane protein adhesins (VompA and VompB), peptidyl-prolyl cis-trans-isomerase (PpI), and hemin-binding protein E (HbpE) were recognized most frequently by sera from patients, which is consistent with surface expression of these virulence factors during human infection.