Bartonella bacilliformis is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrión’s disease. After constructing a low-passage GFP-labeled strain of B. bacilliformis, we artificially infected Lutzomyia verrucarum and L. longipalpis populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72h when B. bacilliformis was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in L. verrucarum, but colonization of L. longipalpis was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of B. bacilliformis in L. longipalpis was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that L. longipalpis could potentially serve as a vector. The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.
In general, Bartonella infections are zoonooses and are transmitted to humans by hematophagous (blood eating) arthropods such as fleas, lice, ticks, mites and sand flies. Bartonella bacilliformis is found only in Andean regions of South America and is transmitted to humans by a few select species of sand flies, resulting in Carrión’s disease. Our current understanding is that Carrión’s disease is not a zoonosis, as an animal reservoir has not been identified. In this study, we analyzed B. bacilliformis colonization in two species of sand flies in an attempt to understand why Lutzomyia verrucarum is a competent vector and Lutzomyia longipalpis is incapable of transmitting this pathogen between humans. We found that B. bacilliformis remains in the abdominal midgut of the non-competent vector and is progressively digested until no viable bacteria remain (7d). Furthermore, large groups of L. longipalpis were used to demonstrate that this non-competent species is incapable of mechanically transmitting viable bacteria between artificial blood meals simply by contaminated mouthparts. In L. verrucarum, B. bacilliformis colonizes the lumen of the digestive tract beyond the intra-peritrophic space in large numbers and persists (>14d).
Bartonella species are fastidious, gram negative bacteria, some of which are transmitted by arthropod vectors, including fleas, sandflies, and lice. There is very little information regarding the interaction and/or transmission capabilities of Bartonella species by ticks. In the present study, we demonstrate successful infection of the Amblyomma americanum cell line, AAE12, by seven Bartonella isolates and three Candidatus Bartonella species by electron or light microscopy. With the exception of Bartonella bovis, infection with all other examined Bartonella species induced cytopathic effects characterized by heavy cellular vacuolization and eventually cell lysis. Furthermore, using quantitative real time PCR (qPCR), we demonstrated significant amplification of two B. henselae genotype I isolates in the A. americanum cell line over a 5 days period. Ultimately, tick-cell derived Bartonella antigens may prove useful for the development of more sensitive diagnostic reagents and may assist in the development of an effective vaccine to prevent the further spread of disease caused by these organisms.
Bartonella species; Tick cell lines; Bacteria; Replication; Cell culture
Bartonella bacilliformis is the bacterial agent of Carrión's disease and is presumed to be transmitted between humans by phlebotomine sand flies. Carrión's disease is endemic to high-altitude valleys of the South American Andes, and the first reported outbreak (1871) resulted in over 4,000 casualties. Since then, numerous outbreaks have been documented in endemic regions, and over the last two decades, outbreaks have occurred at atypical elevations, strongly suggesting that the area of endemicity is expanding. Approximately 1.7 million South Americans are estimated to be at risk in an area covering roughly 145,000 km2 of Ecuador, Colombia, and Peru. Although disease manifestations vary, two disparate syndromes can occur independently or sequentially. The first, Oroya fever, occurs approximately 60 days following the bite of an infected sand fly, in which infection of nearly all erythrocytes results in an acute hemolytic anemia with attendant symptoms of fever, jaundice, and myalgia. This phase of Carrión's disease often includes secondary infections and is fatal in up to 88% of patients without antimicrobial intervention. The second syndrome, referred to as verruga peruana, describes the endothelialcell-derived, blood-filled tumors that develop on the surface of the skin. Verrugae are rarely fatal, but can bleed and scar the patient. Moreover, these persistently infected humans provide a reservoir for infecting sand flies and thus maintaining B. bacilliformis in nature. Here, we discuss the current state of knowledge regarding this life-threatening, neglected bacterial pathogen and review its host-cell parasitism, molecular pathogenesis, phylogeny, sand fly vectors, diagnostics, and prospects for control.
Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated (≥2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 5′ UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription.
Coxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, ∼7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. blast analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni2+ or Zn2+, and roughly halved in the presence of Co2+ or Fe3+. The limB gene was maximally expressed at 3–7 days post-infection in Coxiella-infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (∼7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (∼8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.
Borrelia hermsii, a causative agent of relapsing fever of humans in western North America, is maintained in enzootic cycles that include small mammals and the tick vector Ornithodoros hermsi. In mammals, the spirochetes repeatedly evade the host’s acquired immune response by undergoing antigenic variation of the variable major proteins (Vmps) produced on their outer surface. This mechanism prolongs spirochete circulation in blood, which increases the potential for acquisition by fast-feeding ticks and therefore perpetuation of the spirochete in nature. Antigenic variation also underlies the relapsing disease observed when humans are infected. However, most spirochetes switch off the bloodstream Vmp and produce a different outer surface protein, the variable tick protein (Vtp), during persistent infection in the tick salivary glands. Thus the production of Vmps in mammalian blood versus Vtp in ticks is a dominant feature of the spirochete’s alternating life cycle. We constructed two mutants, one which was unable to produce a Vmp and the other was unable to produce Vtp. The mutant lacking a Vmp constitutively produced Vtp, was attenuated in mice, produced lower cell densities in blood, and was unable to relapse in animals after its initial spirochetemia. This mutant also colonized ticks and was infectious by tick-bite, but remained attenuated compared to wild-type and reconstituted spirochetes. The mutant lacking Vtp also colonized ticks but produced neither Vtp nor a Vmp in tick salivary glands, which rendered the spirochete noninfectious by tick bite. Thus the ability of B. hermsii to produce Vmps prolonged its survival in blood, while the synthesis of Vtp was essential for mammalian infection by the bite of its tick vector.
Borrelia hermsii, an agent of tick-borne relapsing fever when infecting humans, employs antigenic variation of the variable major proteins (Vmps) to escape the host immune response. This mechanism allows the bacteria to persist in the blood of a mammal, which increases their potential for acquisition by their tick vector Ornithodoros hermsi. Once in the tick, the bacteria move from the midgut to salivary glands where the Vmps are replaced with another major surface protein, the variable tick protein (Vtp). We constructed two mutants, one that was unable to produce a Vmp (Vmp−) and another that was unable to produce Vtp (Δvtp). The Vmp− mutant could not reach as high bacterial levels in the blood of mice when infected by needle-inoculation and tick bite compared to the parent strain, and was incapable of relapsing. The Δvtp mutant was able to colonize ticks, but was non-infectious by tick bite. Our study provides insight into the roles of the Vmps and Vtp in the infectivity of B. hermsii by showing the importance of antigenic variation for prolonging bacteria levels in the host as well as the requirement of Vtp for mammalian infection by the bite of its tick vector.
Borrelia burgdorferi, the causative agent of Lyme disease, cycles in nature between a vertebrate host and a tick vector. We demonstrate that B. burgdorferi can utilize several sugars that may be available during persistence in the tick, including trehalose, N-acetylglucosamine (GlcNAc) and chitobiose. The spirochete grows to a higher cell density in trehalose, which is found in tick hemolymph, than in maltose; these two disaccharides differ only in the glycosidic linkage between the glucose monomers. Additionally, B. burgdorferi grows to a higher density in GlcNAc than in the GlcNAc dimer chitobiose, both of which may be available during tick molting. We have also investigated the role of malQ (bb0166), which encodes an amylomaltase, in sugar utilization during the enzootic cycle. In other bacteria, MalQ is involved in utilizing maltodextrins and trehalose, but we show that, unexpectedly, it is not needed for B. burgdorferi to grow in vitro on any of the sugars assayed. In addition, infection of mice by needle inoculation or tick bite, as well as acquisition and maintenance of the spirochete in the tick vector, does not require MalQ.
Lyme disease; carbohydrate; amylomaltase; trehalose; Ixodes
Coxiella burnetii is the bacterial agent of Q fever in humans. Acute Q fever generally manifests as a flu-like illness and is typically self-resolving. In contrast, chronic Q fever usually presents with endocarditis and is often life-threatening without appropriate antimicrobial therapy. Unfortunately, available options for the successful treatment of chronic Q fever are both limited and protracted (>18 months). Pentamidine, an RNA splice inhibitor used to treat fungal and protozoal infections, was shown to reduce intracellular growth of Coxiella by ca. 73% at a concentration of 1 μM (ca. 0.6 μg/mL) compared with untreated controls, with no detectable toxic effects on host cells. Bacterial targets of pentamidine include Cbu.L1917 and Cbu.L1951, two group I introns that disrupt the 23S rRNA gene of Coxiella, as demonstrated by the drug's ability to inhibit intron RNA splicing in vitro. Since both introns are highly conserved among all eight genotypes of the pathogen, pentamidine is predicted to be efficacious against numerous strains of C. burnetii. To our knowledge, this is the first report describing antibacterial activity for this antifungal/antiprotozoal agent.
Coxiella; Pentamidine; Group I intron; RNA splicing
Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen's proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100°C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain's ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P < 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen's early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella's metabolism during intracellular replication.
It has been nearly two decades since the discovery of Bartonella as an agent of bacillary angiomatosis in AIDS patients and persistent bacteremia and ‘nonculturable’ endocarditis in homeless people. Since that time, the number of Bartonella species identified has increased from one to 24, and 10 of these bacteria are associated with human disease. Although Bartonella is the only genus that infects human erythrocytes and triggers pathological angiogenesis in the vascular bed, the group remains understudied compared with most other bacterial pathogens. Numerous questions regarding Bartonella's molecular pathogenesis and epidemiology remain unanswered. Virtually every mammal harbors one or more Bartonella species and their transmission typically involves a hematophagous arthropod vector. However, many details regarding epidemiology and the public health threat imposed by these animal reservoirs is unclear. A handful of studies have shown that bartonellae are highly-adapted pathogens whose parasitic strategy has evolved to cause persistent infections of the host. To this end, virulence attributes of Bartonella include the subversion of host cells with effector molecules delivered via a type IV secretion system, induction of pathological angiogenesis through various means, including inhibition of apoptosis and activation of hypoxia-inducing factor 1, use of afimbrial adhesins that are orthologs of Yersinia adhesin A, incorporation of lipopolysaccharides with low endotoxic potency in the outer membrane, and several other virulence factors that help Bartonella infect and persist in erythrocytes and endothelial cells of the host circulatory system.
angiogenesis; Bartonella; bartonellosis; hemotrophy; infection; virulence
We hypothesize a potential role for Borrelia burgdorferi OspC in innate immune evasion at the initial stage of mammalian infection. We demonstrate that B. burgdorferi is resistant to high levels (>200 μg/ml) of cathelicidin and that this antimicrobial peptide exhibits limited binding to the spirochetal outer membrane, irrespective of OspC or other abundant surface lipoproteins. We conclude that the essential role of OspC is unrelated to resistance to this component of innate immunity.
Bartonella quintana is a gram-negative agent of trench fever, chronic bacteremia, endocarditis, and bacillary angiomatosis in humans. B. quintana has the highest known hemin requirement among bacteria, but the mechanisms of hemin acquisition are poorly defined. Genomic analyses revealed a potential locus dedicated to hemin utilization (hut) encoding a putative hemin receptor, HutA; a TonB-like energy transducer; an ABC transport system comprised of three proteins, HutB, HutC, and HmuV; and a hemin degradation/storage enzyme, HemS. Complementation analyses with Escherichia coli hemA show that HutA functions as a hemin receptor, and complementation analyses with E. coli hemA tonB indicate that HutA is TonB dependent. Quantitative reverse transcriptase PCR analyses show that hut locus transcription is subject to hemin-responsive regulation, which is mediated primarily by the iron response regulator (Irr). Irr functions as a transcriptional repressor of the hut locus at all hemin concentrations tested. Overexpression of the ferric uptake regulator (fur) represses transcription of tonB in the presence of excess hemin, whereas overexpression of the rhizobial iron regulator (rirA) has no effect on hut locus transcription. Reverse transcriptase PCR analyses show that hutA and tonB are divergently transcribed and that the remaining hut genes are expressed as a polycistronic mRNA. Examination of the promoter regions of hutA, tonB, and hemS reveals consensus sequence promoters that encompass an H-box element previously shown to interact with B. quintana Irr.
The Lyme disease spirochete Borrelia burgdorferi alters the expression of outer surface protein (osp) genes as the bacterium cycles between ticks and mammals. OspA is produced as borreliae enter the tick vector and remains a major surface antigen during midgut colonization. To elucidate the role of OspA in the vector, we created an insertional deletion of ospA in strain B31-A3. The ospA mutant infects mice when it is injected intradermally and is acquired by larval ticks fed on these mice, where it persists through the molt to the nymph stage. Bacterial survival rates in artificially infected tick larvae fed on naïve mice were compared with those in the vector fed on immune mice. The ospA mutant proliferates in larvae if it is exposed to blood from naïve mice, but it declines in density after larval feeding if the blood is from immune mice. When uninfected larvae are fed on B-cell-deficient mice infected with the ospA mutant, larvae show borrelial densities and persistence that are significantly greater than those fed on infected, immunocompetent mice. We conclude that OspA serves a critical antibody-shielding role during vector blood meal uptake from immune hosts and is not required for persistence in the tick vector.
We previously identified a five-member family of hemin-binding proteins (Hbp's) of Bartonella quintana that bind hemin on the outer surface but share no homology with known bacterial heme receptors. Subsequently, we demonstrated that expression of the hbp family is significantly influenced by oxygen, heme, and temperature conditions encountered by the pathogen in the human host and the body louse vector; e.g., we observed a dramatic (>100-fold) increase in hbpC transcript levels in response to the “louse-like” temperature of 30°C. The goal of the present study was to identify a transcription factor(s) involved in the coordinated and differential regulation of the hbp family. First, we used quantitative real-time PCR (qRT-PCR) to show that the same environmental conditions generate parallels in the transcript profiles of four candidate transcriptional regulators (Irr, Fur, RirA, and BatR) described in the order Rhizobiales, with the greatest overall change in the transcription of irr (a >5-fold decrease) at a “louse-like” temperature, suggesting that Irr may function as an hbpC repressor. Second, a B. quintana strain hyperexpressing Irr was constructed; it exhibits a “bloodstream-like” hbp transcript profile in the absence of an environmental stimulus (i.e., hbpC is repressed and hbpA, hbpD, and hbpE mRNAs are relatively abundant). Furthermore, when this strain is grown at a “louse-like” temperature, an inversion of the transcript profile occurs, where derepression of hbpC and repression of hbpA, hbpD, and hbpE are readily evident, strongly suggesting that Irr and temperature influence hbp family expression. Third, electrophoretic mobility shift analyses show that recombinant Irr binds specifically to the hbpC promoter region at a sequence that is highly conserved in Bartonella hbp genes, which we designated the hbp family box, or “H-box.” Fourth, we used the H-box to search the B. quintana genome and discovered a number of intriguing open reading frames, e.g., five members of a six-member family of cohemolysin autotransporters. Finally, qRT-PCR data regarding the effects of Fur and RirA overexpression on the hbp family are provided; they show that Fur's effect on the hbp family is relatively minor but RirA generates a “bloodstream-like” hbp transcript profile in the absence of an environmental stimulus, as observed for the Irr-hyperexpressing strain.
The groESL operon of Bartonella bacilliformis, a facultative intracellular, Gram-negative bacterium and etiologic agent of Oroya Fever, was characterized. Sequence analysis revealed an operon containing two genes of 294 (groES) and 1632 nucleotides (groEL) separated by a 55-nt intergenic spacer. The operon is preceded by a 72-nt ORF (ORF1) that encodes a hypothetical protein with homology to a portion of the HrcA repressor for groESL. A divergent fumarate hydratase C (fumC) gene lies further upstream. Deduced amino acid sequences for B. bacilliformis GroEL and GroES revealed a high degree of identity with homologues from other Bartonella and α-Protebacteria. A single transcriptional start site (TSS) was mapped 79 nucleotides upstream of the groES start codon, regardless of incubation temperature. The TSS was located immediately 5′ to a potential controlling inverted repeat of chaperonin expression (CIRCE) element and is preceded by a σ70-like promoter. The operon is followed by a predicted rho-independent transcriptional terminator. Northern blot analysis indicated that groES and groEL are co-transcribed as a single mRNA of ∼2.4 kb. A 6-h time course analysis by qRT-PCR showed that groEL expression increases 1.3-fold within 30 min of a temperature upshift from 30 to 37 °C, with maximum transcription reached after 60 min (∼4.3-fold), followed by a steady decrease to background (30 °C) transcription levels by 6 h. Western blot analysis revealed a 1.4- and 1.5-fold increase in GroEL synthesis following a temperature upshift or by inhibiting DNA supercoiling with coumermycin A1, respectively. Functional expression and complementation of temperature-sensitive Escherichia coli groES or groEL mutants with the cloned operon allowed them to grow at otherwise restrictive temperatures.
GroESL operon; Bartonella; Heat shock protein; Chaperonin; GroEL; GroES
Of all bacteria, Bartonella quintana has the highest reported in vitro hemin requirement, yet an explanation for this remains elusive. To produce diseases such as trench fever, endocarditis, and bacillary angiomatosis, B. quintana must survive and replicate in the disparate environments of the Pediculus humanus corporis (body louse) gut and the human vasculature. We previously identified a five-member family of hemin binding proteins (Hbps) synthesized by B. quintana that bind hemin on the outer surface but share no similarity to known bacterial heme receptors. In the present study, we examine the transcription, regulation, and synthesis of this virulence factor family by cultivation of the bacterium in environments that simulate natural heme, oxygen, and temperature conditions encountered in the host and insect vector. First, quantitative real-time PCR data show that hbpC expression is regulated by temperature, where a >100-fold increase in transcript quantity was seen at 30°C relative to 37°C, suggesting that HbpC synthesis would be greatest in the cooler temperature of the louse. Second, cultivation at human bloodstream oxygen concentration (5% relative to 21% atmospheric) significantly decreases the transcript quantity of all hbp genes, indicating that expression is influenced by O2 and/or reactive oxygen species. Third, a differential expression pattern within the hbp family is revealed when B. quintana is grown in a range of hemin concentrations: subgroup I (hbpC and hbpB) predominates in a simulated louse environment (high heme), and subgroup II (hbpA, hbpD, and hbpE) is preferentially expressed in a simulated human background (low heme). By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and matrix-assisted laser desorption ionization—time of flight mass spectrometry fingerprinting, we demonstrate that synthesis of HbpA correlates with hbpA transcript increases observed at low hemin concentrations. Finally, an hbpA promoter-lacZ reporter construct in B. quintana demonstrates that a transcriptional regulator(s) is controlling the expression of hbpA through a cis-acting regulatory element located in the hbpA promoter region.
Relapsing-fever spirochetes achieve high cell densities (>108/ml) in their host's blood, while Lyme disease spirochetes do not (<105/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood. Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B. hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids. Sequence analysis of B. hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B. burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.
Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kanr clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.
This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of ∼77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants of B. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124→Ser), Arg184→Gln, and Thr214→Ala or Thr214→Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E. coli, Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.