In silico identification of drug targets and vaccine candidates against Bartonella quintana: a subtractive proteomics approach.

BACKGROUND: The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite is Bartonella quintana, a Gram-negative, intracellular pathogen that causes bartonellosis in mammalian hosts. OBJECTIVE: Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to B. quintana. METHODS AND FINDINGS: To explore these aspects, we have adopted a subtractive proteomics approach to analyse the proteome of B. quintana. By subtractive proteins between the host and parasite proteome, a set of proteins that are likely unique to the parasite but absent in the host were identified. This analysis revealed that out of the 1197 protein sequences of the parasite, 660 proteins are non-homologous to the human host. Further analysis using the Database of Essential Genes predicted 159 essential proteins, with 28 of these being unique to the pathogen and predicted as potential putative targets. Subcellular localisation of the predicted targets revealed 13 cytoplasmic, eight membranes, one periplasmic, and multiple location proteins. The three-dimensional structure and B cell epitopes of the six membrane antigenic protein were predicted. Four B cell epitopes in KdtA and mraY proteins, three in lpxB and BQ09550, whereas the ftsl and yidC proteins were located with eleven and six B cell epitopes, respectively. MAINS CONCLUSIONS: This insight prioritises such proteins as novel putative targets for further investigations on their potential as drug and vaccine candidates.

Systemic Bartonellosis Manifesting With Endocarditis and Membranoproliferative Glomerulonephritis.

Cat scratch disease caused by Bartonella species is mostly benign and self-limiting condition. Systemic infection is uncommon in immunocompetent host. We describe the case of a 66-year-old male who presented with sudden painless left eye blindness and brown-colored urine. Laboratory findings revealed progressively rising serum creatinine in association with nephrotic-range proteinuria at 7 g/day and glomerular hematuria on urinalysis. An echocardiogram demonstrated mitral and tricuspid valve vegetations despite multiple negative blood cultures. The left eye blindness was attributed to retinal artery occlusion from septic valvular embolus. Kidney biopsy showed membranoproliferative glomerulonephritis pattern of injury with "full house" pattern on immunofluorescent staining with subendothelial deposits on electron microscopy. Markedly elevated IgG (immunoglobulin G) titers for B henselae and B quintana were discovered. The patient had several cats at home. Kidney failure rapidly progressed to require hemodialysis. Once the diagnosis of systemic bartonellosis was confirmed, doxycycline (for 4 months) with rifampicin (for 3 months) were initiated. Repeat echocardiogram in 4 months demonstrated a resolution of valvular vegetations; however, the left eye blindness was permanent. In the present case the correct diagnosis of systemic bartonellosis allowed institution of appropriate antibiotic therapy and to also achieve a partial recovery of renal function and to discontinue hemodialysis.

Unusual subdural empyema in a homeless patient diagnosed by molecular approach: a case report.

BACKGROUND: We report a case of subdural empyema in a homeless patient caused by Bartonella quintana. B. quintana is a facultative intracellular bacteria for which bacterial growth is fastidious. The molecular biology approach has been a real help in establishing the diagnosis. CASE REPORT: A 59-years old homeless patient, with a history of chronic alcohol abuse, was brought to the emergency department with a massive subdural empyema. Extensive microbiological evaluation didn't reveal any pathogen in the pus collected before antibiotic treatment. B. quintana was detected in the pus from the empyema using a 16S rRNA-based PCR. Histology of intraoperative samples was consistent with the diagnosis and a serological assay was positive. The patient responded well to a treatment that included craniectomy with drainage of the loculated pus, total removal of the infected capsule and a combination of antibiotics. CONCLUSION: This unique case of B. quintana-related empyema illustrates the risk of secondary infection of subdural hematoma with B. quintana since such infections have recently reemerged, predominantly among the homeless populations. Patients with subdural empyema in at-risk populations should be systematically evaluated for B. quintana with an appropriate diagnostic approach involving molecular biology.

Evaluation of cell culture-grown Bartonella antigens in immunofluorescent antibody assays for the serological diagnosis of bartonellosis in dogs.

BACKGROUND: Because of poor sensitivity and questionable specificity of immunofluorescent antibody assays (IFAs), serological diagnosis of Bartonella species infections in dogs remains challenging. Despite limitations, IFA testing is the historical "gold standard" for Bartonella serodiagnosis in animals and humans. Because most diagnostic laboratories test against only 1 or 2 Bartonella spp., testing against a broader panel of Bartonella antigens may enhance diagnostic sensitivity and specificity. OBJECTIVE: To evaluate the sensitivity and specificity of Bartonella IFA using 8 cell culture-grown Bartonella spp. isolates. ANIMALS: Archived serum samples from 34 Bartonella spp. naturally exposed, polymerase chain reaction (PCR)-positive dogs and from 26 PCR-negative and IFA-negative dogs. METHODS: Bartonella IFA sensitivity and specificity were assessed using cell culture-grown whole cell antigens derived from 3 Bartonella henselae (Bh) strains (Bh Houston 1, Bh San Antonio Type 2, Bh California 1), 3 Bartonella vinsonii subsp. berkhoffii genotypes (Bvb I, II, and III), Bartonella koehlerae (Bk), and Bartonella quintana (Bq). RESULTS: Only 62% of 34 Bartonella spp. PCR-positive dogs were seroreactive to any of the 8 Bartonella IFA antigens, indicating low IFA sensitivity. PCR-positive dogs were most often IFA seroreactive to Bq (n = 15), to Bvb II (n = 13), or to both (n = 9) antigens. Of the 26 previously IFA-negative/PCR-negative dogs, 4 (15%) were seroreactive using the expanded antigen panel. CONCLUSION AND CLINICAL IMPORTANCE: Despite IFA testing of dogs against 8 different Bartonella isolates, IFA sensitivity remained poor, and specificity was only 85%. Development of a reliable serological assay is needed to facilitate the diagnosis of Bartonella infection in dogs.

Bartonella quintana and Typhus Group Rickettsiae Exposure among Homeless Persons, Bogotá, Colombia.

In 2015, we investigated Bartonella quintana and typhus group rickettsiae in body lice from homeless persons in Bogotá, Colombia. We found B. quintana-infected body lice and seroprevalence of this microorganism in 19% of homeless persons and typhus group rickettsiae in 56%. Public health professionals should start preemptive measures and active vector control.

Blood Culture-Negative Endocarditis, Morocco.

We investigated the microorganisms causing blood culture-negative endocarditis (BCNE) in Morocco. We tested 19 patients with BCNE by serologic methods, molecular methods, or both and identified Bartonella quintana, Staphylococcus aureus, Streptococcus equi, and Streptococcus oralis in 4 patients. These results highlight the role of these zoonotic agents in BCNE in Morocco.

BARTONELLA QUINTANA-ASSOCIATED NEURORETINITIS: LONGITUDINAL SPECTRAL-DOMAIN OPTICAL COHERENCE TOMOGRAPHIC FINDINGS.

PURPOSE: To report an unusual case of neuroretinitis caused by Bartonella quintana and its spectral-domain optical coherence tomographic (SD-OCT) features. METHODS: A 12-year-old girl presented with unilateral neuroretinitis with stellate maculopathy. Bartonellosis was confirmed after serologic testing for antibodies to B. quintana. RESULTS: Color photograph of the right eye revealed papillitis and stellate macular exudation. spectral-domain optical coherence tomography of the right eye revealed hyperreflective dots in the outer nuclear and outer plexiform layers, as well as disruption and loss of the external limiting membrane, ellipsoid zone, and interdigitation zone in the foveal area. CONCLUSION: The authors report an unusual case of neuroretinitis by B. quintana and its spectral-domain optical coherence tomographic findings.

Quintessential Culture-Negative Endocarditis.

Bartonella spp are important causes of culture-negative endocarditis, generally causing a subacute insidious form of endocarditis, often leading to a delay in diagnosis. Most patients have fever and often present with signs and symptoms of heart failure. The diagnosis is frequently established only on meticulous examination of the resected heart valve with the polymerase chain reaction technique. We present a case of B quintana mitral and aortic valve endocarditis with associated severe valvular insufficiency and decompensated heart failure precipitated by Streptococcus pneumoniae bacteremia, necessitating urgent surgical valve replacement. Pathologic examination of the valve complemented by serologic and molecular testing established the surprising diagnosis of B quintana endocarditis.

[Seroprevalence of Bartonella henselae and Bartonella quintana in blood donors in Aydin province, Turkey]. / Aydin ili kan donörlerinde Bartonella henselae ve Bartonella quintana seroprevalansi

Bartonella species cause several diseases in humans such as cat stratch disease, bacillary angiomatosis, peliosis hepatis, endocarditis, Carrion disease and trench fever. Cat scratch disease and bacillary angiomatosis cases have already been reported in Turkey. Studies from our region, namely Aydin (a province located at Western Anatolia, Turkey) indicated that mean Bartonella henselae IgG seropositivity rate is 11.5% in risk groups and may reach to 26.5% in pet owners. The aim of this study was to determine the seroprevalence of B.henselae and B.quintana in healthy blood donors in our university hospital in Aydin, for estimating the transmission risk via transfusion. The study was designed as a cross-sectional epidemiological study. A total of 333 samples taken from blood donors (49 female, 284 male) who were sequentially admitted to the blood center of the university hospital, in January 2011 were included in the study. All sera were screened in terms of B.henselae and Bartonella quintana IgG antibodies by using two different indirect immunofluorescent antibody (IFA) commercial kits (Vircell, Spain; Focus, USA). Slides were examined at a final magnification of x400 on fluorescent microscope by two different assigned researchers. Fluorescent intensity was graded between 1+ to 4+, and the samples with fluorescence value of ≥ 2+ were considered as positive. The seropositivity rate of IgG antibodies to B.henselae was found as 3.3% (11/333) in blood donors. This rate was 4.1% in female, and 3.2% in male donors, showing no statistically significant difference between the genders (p= 0.668). B.henselae antibody titers were detected as 1/64 in 6 (1.8%), 1/128 in 4 (1.2%) and 1/1024 in 1 (0.3%) patient. All of the B.henselae IgG positive samples also yielded relatively low positivity for B.quintana IgG, possibly indicating cross reactivity. The fluorescence intensity for different kits used was found to be the same in all but one titer. The results reported by two researchers were found to differ only in the samples graded 1+ or below. However, the evaluation differences between the kits and the researchers did not affect the results. It was concluded that B.henselae infection might be found in the blood donors in our region, thus, a detailed questionnaire prior to blood donation might be helpful to prevent transmission of B.henselae by blood transfusion.

Hemin-binding proteins as potent markers for serological diagnosis of infections with Bartonella quintana.

It is difficult to distinguish infections with different Bartonella species using commercially available immunofluorescence (indirect immunofluorescent antibody [IFA]) assay kits. To identify appropriate proteins for serodiagnosis of Bartonella quintana infections, we investigated the antigenicity of B. quintana proteins using sera from homeless people with high B. quintana IgG titers in IFA assay. These sera reacted strongly to an outer membrane protein, hemin-binding protein D (HbpD). Further, serum from an endocarditis patient infected with B. quintana reacted to HbpB and HbpD. To locate the antigenic sites within the proteins, we generated deletion mutants of HbpB and HbpD. Amino acid residues 89 to 220 of HbpB and 151 to 200 of HbpD were identified as the minimum regions required for recognition by these sera. Several oligopeptides comprising parts of the minimum regions of HbpB and HbpD were synthesized, and their immunoreactivity with the above-mentioned sera was tested by enzyme-linked immunosorbent assay (ELISA). Serum from the endocarditis patient reacted similarly to synthetic peptides HbpB2 (amino acid residues 144 to 173 of HbpB) and HbpD3 (151 to 200 residues of HbpD), while sera from the other subjects reacted to HbpD3. These results indicate that synthetic peptides HbpB2 and HbpD3 might be suitable for developing serological tools for differential diagnosis of B. quintana infections from other Bartonella infections.

Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation.

Bartonella spp. are facultative intracellular bacteria that typically cause a long-lasting intraerythrocytic bacteremia in their mammalian reservoir hosts, thereby favoring transmission by blood-sucking arthropods. In most cases, natural reservoir host infections are subclinical and the relapsing intraerythrocytic bacteremia may last weeks, months, or even years. In this review, we will follow the infection cycle of Bartonella spp. in a reservoir host, which typically starts with an intradermal inoculation of bacteria that are superficially scratched into the skin from arthropod feces and terminates with the pathogen exit by the blood-sucking arthropod. The current knowledge of bacterial countermeasures against mammalian immune response will be presented for each critical step of the pathogenesis. The prevailing models of the still-enigmatic primary niche and the anatomical location where bacteria reside, persist, and are periodically seeded into the bloodstream to cause the typical relapsing Bartonella spp. bacteremia will also be critically discussed. The review will end up with a discussion of the ability of Bartonella spp., namely Bartonella henselae, Bartonella quintana, and Bartonella bacilliformis, to induce tumor-like vascular deformations in humans having compromised immune response such as in patients with AIDS.

High prevalence of Bartonella henselae and Bartonella quintana antibodies in Croatian patients presenting with lymphadenopathy.

Between 2007 and 2010, a total of 268 Croatian patients with lymphadenopathy were tested for IgM/IgG antibodies to Bartonella (B.) henselae and B. quintana. Samples from 44.4% patients showed positive IgG antibodies: 35.8% to B. henselae, 6.7% to B. quintana and 1.9% to both Bartonella species. There was no difference in seropositivity between males and females (47.4% vs. 41.5%). Seroprevalence was high in all age groups (40.4-60.9%). Patients from urban and rural areas showed a similar seroprevalence rate (44.1% vs. 44.8%). Positive IgM antibodies were found in 28.3% patients varying from 17.5% and 37.5% among age groups. Most cases were reported from August to March.

Bartonella infection in immunocompromised hosts: immunology of vascular infection and vasoproliferation.

Most infections by genus Bartonella in immunocompromised patients are caused by B. henselae and B. quintana. Unlike immunocompetent hosts who usually develop milder diseases such as cat scratch disease and trench fever, immunocompromised patients, including those living with HIV/AIDS and posttransplant patients, are more likely to develop different and severe life-threatening disease. This paper will discuss Bartonella's manifestations in immunosuppressed patients and will examine Bartonella's interaction with the immune system including its mechanisms of establishing infection and immune escape. Gaps in current understanding of the immunology of Bartonella infection in immunocompromised hosts will be highlighted.

Q fever endocarditis in Romania: the first cases confirmed by direct sequencing.

Infective endocarditis (IE) is a serious, life-threatening disease with highly variable clinical signs, making its diagnostic a real challenge. A diagnosis is readily made if blood cultures are positive, but in 2.5 to 31% of all infective endocarditis cases, routine blood cultures are negative. In such situations, alternative diagnostic approaches are necessary. Coxiella burnetii and Bartonella spp. are the etiological agents of blood culture-negative endocarditis (BCNE) most frequently identified by serology. The purpose of this study is to investigate the usefulness of molecular assays, as complementary methods to the conventional serologic methods for the rapid confirmatory diagnostic of Q fever endocarditis in patients with BCNE. Currently, detection of C. burnetii by culture or an antiphase I IgG antibody titers >800 represents a major Duke criterion for defining IE, while a titers of >800 for IgG antibodies to either B. henselae or B. quintana is used for the diagnosis of endocarditis due to Bartonella spp. We used indirect immunofluorescence assays for the detection of IgG titers for C. burnetii, B. henselae and B. quintana in 57 serum samples from patients with clinical suspicion of IE. Thirty three samples originated from BCNE patients, whereas 24 were tested before obtaining the blood cultures results, which finally were positive. The results of serologic testing showed that nine out of 33 BCNE cases exhibited antiphase I C. burnetii IgG antibody titer >800, whereas none has IgG for B. henselae or B. quintana. Subsequently, we used nested-PCR assay for the amplification of C. burnetii DNA in the nine positive serum samples, and we obtained positive PCR results for all analyzed cases. Afterwards we used the DNA sequencing of amplicons for the repetitive element associated to htpAB gene to confirm the results of nested-PCR. The results of sequencing allowed us to confirm that C. burnetii is the causative microorganism responsible for BCNE. In conclusion, the nested PCR amplification followed by direct sequencing is a reliable and accurate method when applied to serum samples, and it may be used as an additional test to the serological methods for the confirmatory diagnosis of BCNE cases determined by C. burnetii.

Receptor recognition of and immune intracellular pathways for Veillonella parvula lipopolysaccharide.

Veillonella parvula is an anaerobic gram-negative coccus that is part of the normal flora of the animal and human mouth and gastrointestinal and genitourinary tracts. Oral V. parvula is involved in the development of early periodontal disease as well as different types of serious infections. Present data on molecular mechanisms responsible for innate immune response against Veillonella are very scanty. The aim of this study was to investigate the Toll-like receptor (TLR) pathways responsible for V. parvula lipopolysaccharide (LPS) and to identify the intracellular pathways induced by this recognition. V. parvula LPS stimulated tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) release in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Pretreatment of cells with a TLR4 antagonist significantly reduced TNF-alpha and IL-6 production in PBMC stimulated with either Veillonella or Escherichia coli LPS. However, V. parvula LPS was 10- to 100-fold less active than E. coli LPS for cytokine induction. TNF-alpha, IL-1beta, IL-6, and IL-10 were released in wild-type and TLR2(-/-), but not TLR4(-/-), mouse macrophage cultures. V. parvula LPS was able to activate the human PBMC p38 mitogen-activated protein kinase (MAPK). A specific p38 MAPK inhibitor strongly inhibited V. parvula LPS-induced TNF-alpha, IL-1beta, IL-6, and IL-10. In conclusion, V. parvula LPS is able to induce cytokine production in both human and murine in vitro models, although it is less effective than Enterobacteriaceae LPS. V. parvula LPS-stimulated cytokine induction, as well as p38 MAPK activation, are TLR4-dependent features.

First known case of Bartonella quintana endocarditis in Sweden.

In this report, we present the first known case of Bartonella endocarditis in Sweden. IgG antibody titres to Bartonella spp. were elevated but blood cultures remained negative. Sequencing of a gltA fragment from DNA extracted from heart valve tissue specimens revealed sequence homology with B. quintana.

The Janus face of Bartonella quintana recognition by Toll-like receptors (TLRs): a review.

Bartonella quintana (B. quintana) is a facultative, intracellular bacterium, which causes trench fever, chronic bacteraemia and bacillary angiomatosis. Little is known about the recognition of B. quintana by the innate immune system. In this review, we address the impact of Toll-like receptors (TLRs) on the recognition of B. quintana and the activation of the host defense. When experimental models using human mononuclear cells, transfected CHO cells, or TLR2-/- and TLR4-/- mice were used, differential effects of TLR2 and TLR4 have been observed. B. quintana micro-organisms stimulated cytokine production through TLR2-mediated signals, whereas no role for TLR4 in the recognition of this pathogen was observed. When single, water-phenol extraction was performed, B. quintana LPS, stimulated cytokine production in a TLR2-dependent manner. However, when double extraction was performed in order to generate highly purified LPS, B. quintana LPS entirely lost its capacity to stimulate cytokines, demonstrating that non-LPS components of B. quintana are responsible for the recognition through TLR2. Moreover, B. quintana LPS was shown to be a potent antagonist of Toll-like receptor 4 (TLR4). In conclusion, B. quintana is an inducer of cytokines through TLR2-, but not TLR4-, dependent mechanisms. This stimulation is induced by bacterial components other than lipopolysaccharide. B. quintana LPS is a naturally occurring antagonist of Toll-like receptor 4 (TLR4). In view of the role played by TLR4 in inflammation, B. quintana LPS may be useful as an anti-TLR4 agent with therapeutic potential in both infections and autoimmune inflammation.

Seroprevalence of Bartonella spp. infection in HIV patients in Catalonia, Spain.

BACKGROUND: Although the first clinical descriptions of Bartonella infection were associated with immunocompromised patient with bacillary angiomatosis, we currently know that this organism is directly involved in diseases affecting a large number of patients, regardless of their immune status. Cat scratch disease, hepatic peliosis, and some cases of bacteraemia and endocarditis, are directly caused by some species of the genus Bartonella. The purpose of this study was to determinate the prevalence of IgG antibodies against Bartonella henselae and B. quintana in HIV patients and to identify the epidemiological factors involved. METHODS: Serum samples were collected from HIV patients treated at Hospital de Sabadell. Antibodies to B. henselae and B. quintana from 340 patients were examined by indirect immunofluorescence assay (IFA). Significance levels for univariate statistical test were determined by the Mann-Whitney U test and chi2 test. RESULTS: Of 340 patients, 82 were women and 258 men, with a median age of 42.21 +/- 10.35 years (range 16-86 years). Seventy-six (22.3%) patients reacted with one or more Bartonella antigens. Of all the factors concerning the seroprevalence rate being studied (age, sex, intravenous drugs use, alcohol consumption, CD4 levels, AIDS, HCV, HBV, residential area), only age was statistically significant. CONCLUSION: A high percentage of HIV patients presents antibodies to Bartonella and is increasing with age.

Bartonella quintana lipopolysaccharide is a natural antagonist of Toll-like receptor 4.

Bartonella quintana is a gram-negative microorganism that causes trench fever and chronic bacteremia. B. quintana lipopolysaccharide (LPS) was unable to induce the production of proinflammatory cytokines in human monocytes. Interestingly, B. quintana LPS is a potent antagonist of Toll-like receptor 4 (TLR4), as it inhibited both mRNA transcription and the release of tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and IL-6 by Escherichia coli LPS in human monocytes, at ratios ranging from 1,000:1 to 10:1 (B. quintana LPS to E. coli LPS). Likewise, B. quintana LPS blocked the interaction of E. coli LPS with TLR4 in transfected cell lines. The extent of the inhibitory effect of B. quintana LPS was demonstrated in microarray studies, which showed downregulation of practically all genes induced by LPS in monocytes. Because of the role of TLR4 in inflammation, B. quintana LPS may prove useful as a potent anti-TLR4 agent with therapeutic potential in both infections and autoimmune inflammation.