Bartonella henselae is a zoonotic pathogen that usually causes a self-limiting infection in immunocompetent individuals but often causes potentially life-threatening infections, such as bacillary angiomatosis, in immunocompromised patients. Both diagnosis of infection and research into the molecular mechanisms of pathogenesis have been hindered by the absence of a suitable liquid growth medium. It has been difficult to isolate B. henselae directly from the blood of infected humans or animals or to grow the bacteria in liquid culture media under laboratory conditions. Therefore, we have developed a liquid growth medium that supports reproducible in vitro growth (3-h doubling time and a growth yield of approximately 5 ؋ 10 8 CFU/ml) and permits the isolation of B. henselae from the blood of infected cats. During the development of this medium, we observed that B. henselae did not derive carbon and energy from the catabolism of glucose, which is consistent with genome nucleotide sequence data suggesting an incomplete glycolytic pathway. Of interest, B. henselae depleted amino acids from the culture medium and accumulated ammonia in the medium, an indicator of amino acid catabolism. Analysis of the culture medium throughout the growth cycle revealed that oxygen was consumed and carbon dioxide was generated, suggesting that amino acids were catabolized in a tricarboxylic acid (TCA) cycle-dependent mechanism. Additionally, phage particles were detected in the culture supernatants of stationary-phase B. henselae, but not in mid-logarithmic-phase culture supernatants. Enzymatic assays of whole-cell lysates revealed that B. henselae has a complete TCA cycle. Taken together, these data suggest B. henselae may catabolize amino acids but not glucose to derive carbon and energy from its host. Furthermore, the newly developed culture medium should improve isolation of B. henselae and basic research into the pathogenesis of the bacterium.Bartonella henselae is a medically important gram-negative, zoonotic pathogen. Cats are the reservoir, and infections are generally asymptomatic. B. henselae causes a diverse and emerging disease spectrum in humans, from self-limiting lymphadenopathy to life-threatening conditions such as bacillary angiomatosis. Cat scratch disease (CSD), a generally benign infection characterized by regional lymphadenopathy and persistent fever, is the most-common manifestation of B. henselae infection in humans. CSD occurs in healthy individuals and affects an estimated 24,000 persons a year in the United States (11). In immunocompromised patients, B. henselae causes more-serious infections, including bacillary angiomatosis and bacillary peliosis, which can be fatal when misdiagnosed and improperly treated. A major contributing factor to misdiagnosis is the inherent difficulty in culturing the bacterium. B. henselae takes an average of 21 days to form colonies during primary isolation on blood agar plates, and no reliable liquid growth medium exists (8). In clinical settings, the protracted growth phase ofte...

A hallmark of Bartonella henselae is persistent bacteremia in cats despite the presence of a vigorous host immune response. To understand better the long-term survival of B. henselae in cats, we examined the feline humoral immune response to B. henselae outer membrane (OM) proteins in naturally and experimentally infected cats. Initially, a panel of sera (n ‫؍‬ 42) collected throughout North America from naturally infected cats was used to probe B. henselae total membranes to detect commonly recognized antigens. Twelve antigens reacted with sera from at least 85% of cats, and five were recognized by sera from all cats. To localize these antigens further, OMs were purified on discontinuous sucrose density step gradients. Each membrane fraction (OM, hybrid or inner membrane [IM]) contained less than 1% of the total malate dehydrogenase activity (soluble marker), indicating very little contamination by cytoplasmic proteins. FtsI, an integral IM cell division protein, was used to identify the low-density fraction ( ‫؍‬ 1.13 g/cm 3 ) as putative IM (<5% of the total FtsI localized to the high-density fraction) while lipopolysaccharide (LPS) and Pap31, a homolog of the Bartonella quintana heme-binding protein A (HbpA), defined the high-density fraction ( ‫؍‬ 1.20 g/cm 3 ) as putative OM. Additionally, little evidence of cross-contamination between the IM and OM was evident by two-dimensional gel electrophoresis. When purified OMs were probed with feline sera, antigenic proteins profiles were very similar to those observed with total membranes, indicating that many, but not all, of the immunoreactive proteins detected in the initial immunoblots were OM components. Interestingly, two-dimensional immunoblots indicated that B. henselae LPS and members of the Hbp family of proteins did not appear to stimulate an humoral response in any infected cats. Seven proteins were recognized by at least 70% of sera tested, but only three were recognized by all sera. Nanospray-tandem mass spectrometry was used to identify OM components, including the immunodominant OM proteins. Recognition of the nonimmunogenic nature of the major OM components, such as LPS, and identification of the predominant immunogens should elucidate the mechanisms by which B. henselae establishes persistent bacteremic infections within cats. Additionally, the common antigens may serve as potential feline vaccine candidates to eliminate the pathogen from its animal reservoir.

CbpA is a DnaJ homolog that functions as a DnaK cochaperone. Several cellular processes, including growth at low and high temperatures and septum formation during cell division, require either CbpA or DnaJ. CbpA is encoded in an operon with the gene for CbpM, which is a specific in vivo and in vitro inhibitor of CbpA. Here, we have cooverexpressed CbpA with CbpM in a ⌬cbpAM ⌬dnaJ strain and examined the resulting phenotypes. Under these conditions, sufficient free CbpA activity was present to support growth at low temperatures, but not at high temperatures. Defects in cell division and in replication were also partially complemented by CbpA when cooverexpressed with CbpM. Utilizing reporter fusions, we demonstrated that the cbpAM operon was maximally transcribed at the transition from exponential growth to stationary phase. Transcription was controlled by the S and Lrp global regulators, and both leucine availability and growth temperature influenced transcription. CbpA and CbpM accumulated to similar levels in stationary phase, ϳ2,300 monomers per cell. When not bound to CbpA, CbpM was unstable and was degraded by the Lon and ClpAP proteases. These data demonstrate that CbpA activity is controlled at multiple levels.

CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in T. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn 2؉ while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25°C, retained only 50% activity after incubation for 20 min at 34°C or 10 min at 37°C, and was completely inactive after 10 min at 42°C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42°C resulted in a 7-to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum.Syphilis, a sexually transmitted disease caused by the spirochete Treponema pallidum, remains a major public health problem in the world. T. pallidum cannot be cultivated in vitro, making it difficult to assess the role of genes in physiology, survival in the host, and pathogenesis. One approach to studying the functions of T. pallidum genes is to clone and overexpress these genes in Escherichia coli and then characterize the recombinant proteins in vitro. This approach was taken recently to characterize the TroR regulatory protein from T. pallidum (28). In the presence of Mn 2ϩ , TroR binds the operator of the transport-related operon (tro) and represses transcription. The tro operon contains six genes (14). The first four genes encode a putative ABC metal transport system (troA to -D), the fifth gene encodes TroR (troR), and the last gene encodes a glycolytic enzyme, 3-phosphoglycerate mutase (gpm, referred to as pgm in the T. pallidum genome database), which converts 3-phosphoglycerate (3-PGA) to 2-phosphoglycerate (2-PGA) (8, 11). Since T. pallidum can only generate ATP via glycolysis, 3-phosphoglycerate mutase is a key enzyme for the spirochete.Bacterial phosphoglycerate mutases are divided into two classes, based on their requirement for the cofactor 2,3-diphosphoglycerate (DPG) (10). Phosphoglycerate mutases from spore-forming Bacillus species, such as Bacillus megaterium, Bacillus subtilis, and Bacillus stearothermophilus, are DPG independent b...