Binding phospholipid is a simple, yet flexible, strategy for anchorage of bacterial effectors at cell membrane to manipulate host signaling responses. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate are the only two phospholipid species known to direct bacterial effectors to establish inner leaflet localization at the plasma membrane. Here, selectivity of phosphatidic acid (PA) by bacterial effectors for the plasma membrane anchorage and its molecular entity was identified. C-terminal BID domain of Bartonella T4SS effectors (Beps) directed the plasma membrane localization of Beps in host cells through binding with PA. A hydrophobic segment of the ‘HOOK’ subdomain from BID is inserted into the bilayer to enhance the interaction of positively charged residues with the lipid headgroups. Mutations of a conserved arginine facilitating the electrostatic interaction, a conserved glycine maintaining the stability of the PA binding groove, and hydrophobic residues determining membrane insertion, prevented the anchorage of Beps at the plasma membrane. Disassociation from plasma membrane to cytosol attenuated the BepC capacity to induce stress fiber formation and cell fragmentation in host cells. The substitution of alanine with aspartic acid at the -1 position preceding the conserved arginine residue hindered BepD anchoring at the plasma membrane, a vital prerequisite for its ability to elicit IL-10 secretion in host macrophages. In conclusion, our findings reveal the PA-binding properties of bacterial effectors to establish plasma membrane localization and will shed light on the intricate mechanisms employed by bacterial effectors within host cells.

A 75-year-old man with a history of an untreated hepatitis C virus infection presented with a 4-day history of a pruritic rash on his lower legs. He noted generalized malaise and an unintentional weight loss of 7 kg over the past 2 months. A video showing aortic vegetation is available at NEJM.org.

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 ( f ilamentation i nduced by c -AMP) domain and C-terminus BID ( B artonella i ntracellular d elivery) 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 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.

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.

Human body and head lice are highly related haematophagous ectoparasites but only the body louse has been shown to transmit Bartonella quintana, the causative agent of trench fever. The mechanisms by which body lice became a vector for B. quintana, however, are poorly understood. Following oral challenge, green fluorescent protein-expressing B. quintana proliferated over 9 days postchallenge with the number of bacteria being significantly higher in whole body vs. head lice. The numbers of B. quintana detected in faeces from infected lice, however, were approximately the same in both lice. Nevertheless, the viability of B. quintana was significantly higher in body louse faeces. Comparison of immune responses in alimentary tract tissues revealed that basal transcription levels of peptidoglycan recognition protein and defensins were lower in body lice and the transcription of defensin 1 was up-regulated by oral challenge with wild-type B. quintana in head but not in body lice. In addition, the level of cytotoxic reactive oxygen species generated by epithelial cells was significantly lower in body lice. Although speculative at this time, the reduced immune response is consistent with the higher vector competence seen in body vs. head lice in terms of B. quintana infection.

SUMMARY Bartonellae are blood- and vector-borne Gram-negative bacteria, recognized as emerging pathogens. Whole-blood samples were collected from 58 free-ranging lions (Panthera leo) in South Africa and 17 cheetahs (Acinonyx jubatus) from Namibia. Blood samples were also collected from 11 cheetahs (more than once for some of them) at the San Diego Wildlife Safari Park. Bacteria were isolated from the blood of three (5%) lions, one (6%) Namibian cheetah and eight (73%) cheetahs from California. The lion Bartonella isolates were identified as B. henselae (two isolates) and B. koehlerae subsp. koehlerae. The Namibian cheetah strain was close but distinct from isolates from North American wild felids and clustered between B. henselae and B. koehlerae. It should be considered as a new subspecies of B. koehlerae. All the Californian semi-captive cheetah isolates were different from B. henselae or B. koehlerae subsp. koehlerae and from the Namibian cheetah isolate. They were also distinct from the strains isolated from Californian mountain lions (Felis concolor) and clustered with strains of B. koehlerae subsp. bothieri isolated from free-ranging bobcats (Lynx rufus) in California. Therefore, it is likely that these captive cheetahs became infected by an indigenous strain for which bobcats are the natural reservoir.

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.

ABSTRACT 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.

Human head and body lice are obligatory hematophagous ectoparasites that belong to a single species, Pediculus humanus. Only body lice, however, are vectors of the infectious Gram-negative bacterium Bartonella quintana. Because of their near identical genomes, yet differential vector competence, head and body lice provide a unique model system to study the gain or loss of vector competence. Using our in vitro louse-rearing system, we infected head and body lice with blood containing B. quintana in order to detect both differences in the proliferation of B. quintana and transcriptional differences of immune-related genes in the lice. B. quintana proliferated rapidly in body lice at 6 days post-infection, but plateaued in head lice at 4 days post-infection. RNAseq and quantitative real-time PCR validation analyses determined gene expression differences. Eight immunoresponse genes were observed to be significantly different with many associated with the Toll pathway: Fibrinogen-like protein, Spaetzle, Defensin 1, Serpin, Scavenger receptor A and Apolipoporhrin 2. Our findings support the hypothesis that body lice, unlike head lice, fight infection from B. quintana only at the later stages of its proliferation.

This chapter provides an introduction to the rapidly expanding but still very limited knowledge about the interactions of these Bartonella species and their reservoirs and vectors and the adaptations these bacteria have developed to maintain persistent infection in the host mammalian species. There are now at least 13 named species and probably an equal number of unnamed species. Of these, only four have been definitively associated with human disease: B. henselae, B. quintana, B. bacilliformis, and B. elizabethae. B. quintana infection may be asymptomatic or may be characterized by high fever, severe shin pain, and relapsing symptoms over weeks to months. The species most commonly associated with endocarditis in humans is B. quintana, and most patients with B. endocarditis require replacement of the infected cardiac valve. Granulomatous inflammatory disease (CSD), most commonly of the lymph nodes, occurs in immunocompetent humans infected with B. henselae. In humans and cats, B. quintana and B. henselae bacteremia, respectively, persist despite the development of a humoral antibody response. The study of Bartonella pathogenesis is still in its infancy, and although there is a tractable system for genetic manipulation (via conjugation), it will be essential to identify and characterize virulence factors. The availability of the B. henselae genome sequence in the near future will greatly enhance these studies, and the next phase of comparative genomics and microarrays will provide further insight into the mechanisms of pathogenesis and persistence of B. bacilli.

Bartonella species have become recognized as substantial human pathogens associated with a wide spectrum of diseases. Four members of the genus are known to cause human infection: Bartonella bacillifarmis, B. henselae, B. quintana, and B. elizabethae. Bacillary peliosis hepatis (BP) is a different vasculoproliferative histopathological entity that occurs in the liver and spleen and occasionally in lymph nodes. Patients with BP can develop thrombocytopenia or pancytopenia and elevated levels of hepatic enzymes, most commonly increased alkaline phosphatase. Bartonella can be isolated directly from the cutaneous lesions of bacillary angiomatosis (BA), but recovery is even more difficult and may require cultivation of biopsied tissue with endothelial cells for weeks. It is likely that cat scratch disease (CSD) is the most commonly recognized Bartonella infection of humans, affecting at least 22,000 people each year and costing more than $12 million annually for diagnosis and treatment in the United States alone. The identification of B. henselae as the agent of CSD and the ability to culture this organism and detect antibodies to it have better defined the spectrum of CSD infection. As with other Bartonella species, the transmission of these four Bartonella species among small mammals is presumed to occur via arthropod vectors. Infection in immunocompromised hosts is associated with an unusual stimulation of vascular proliferation (BA), while infection with the same species, B. hensetae, causes an entirely different histopathological response (CSD) in immunocompetent hosts.

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.

ABSTRACT 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.

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 105 colony-forming units [CFU]/ml) and vector (more than 108 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.

ABSTRACT 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 Δ rpoE Δ nepR 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.

Bacillary angiomatosis (BA) was first described by Stoler and colleagues in 1983 in an HIV-infected patient with multiple subcutaneous nodules. Numerous bacilli were observed by Warth in-Starry staining of the biopsicd nodules, and the subcutaneous masses resolved during erythromycin therapy. Subsequently, the BA bacilli visualized using the Warthin-Stilrry silver stain werc noted to have an appearance similar to that of the cat scratch disease (CSD) bacillus.:!') The BA bacillus remained refractory to isolCltion attempts for many years, impeding identification efforts. Studies of bacterial DNA extracted from BA lesions subsequently identified the bacillus as closely related to Bartollclla (Rochalimaea) qllinlmza,4 and after isolation of the bacillus from the blood of hvo HIV-infected patients without SA,' the organism was further characterized and named B. !Iel/selae in 1992.'; The Bartol1ella genus has expanded from a single species in 1993 to >20 species. The BA bacillus was directly cultivated from cutaneous BA lesions for the first time in 1992, which led to the identification of two species of the genus Bartol/tila as causative agents of SA: B. IU!Jlsdae or B. quiutlJJla.· To date, the Bartoella species causing BA has been identified in more than 60 AIDS patients; in all these cases, only two species have been found to cause BA or bacillary peliosis hepatis.M,9 Interestingly, the two different species differ in the predilection to form a specific type of lesion. Bartouella hel/selae, but never B. quilltaua, has been associated with peliosis of the liver or spleen, or both.'} Bartoella htllselnc illso is ilssociilted with lymphadenopathy, and B. quilltal1a with subcutilneous nodules in late stage HIV infection.':I In patients with severe immunosuppression due to HIV infection, organ transplantation or chemotherapy, infection with B. hcnse/ac or B. quintal/a can produce unique vascular proliferative lesions known as BA.IO,1l BA occurs as a late manifestation of HIV infection; in a study of 42 patients with BA, the medirm CD4 lymphocyte count was 21 cells/mm~.12 These vtlsculnr proliferative lesions cnn form in many different organs, including skin, bone, brain parenchyma, lymph nodes, bone marrow and gastrointestinal and respiratory tract. A histopathologically different vascul

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.