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1.  Sequestration and Scavenging of Iron in Infection 
Infection and Immunity  2013;81(10):3503-3514.
The proliferative capability of many invasive pathogens is limited by the bioavailability of iron. Pathogens have thus developed strategies to obtain iron from their host organisms. In turn, host defense strategies have evolved to sequester iron from invasive pathogens. This review explores the mechanisms employed by bacterial pathogens to gain access to host iron sources, the role of iron in bacterial virulence, and iron-related genes required for the establishment or maintenance of infection. Host defenses to limit iron availability for bacterial growth during the acute-phase response and the consequences of iron overload conditions on susceptibility to bacterial infection are also examined. The evidence summarized herein demonstrates the importance of iron bioavailability in influencing the risk of infection and the ability of the host to clear the pathogen.
doi:10.1128/IAI.00602-13
PMCID: PMC3811770  PMID: 23836822
2.  Differential expression of the invasion-associated locus B (ialB) gene of Bartonella bacilliformis in response to environmental cues 
Microbial pathogenesis  2003;34(4):179-186.
Bartonella bacilliformis is the causative agent of the biphasic human disease, Oroya fever. During the primary disease phase, up to 100% of the circulating erythrocytes can be parasitized and 80% lysed. During the secondary phase of this disease, bacterial invasion shifts to endothelial cells lining the vasculature. B. bacilliformis is transferred between human hosts by the sandfly, Lutzomyia verrucarum. To investigate the regulation of ialB by environmental cues signaling vector-to-host transmission; nuclease protection assays were performed to compare the amount of ialB mRNA in bacteria subjected to temperature shift, pH change, oxidative stress, or hemin limitation. The amount of ialB mRNA increased by 223–310% in acid-treated samples and decreased by 28–39% in base-treated samples as compared to bacteria kept at pH 7.2. B. bacilliformis samples showed a 56–63% and 74–80% decrease in ialB mRNA when shifted to 37 °C from growth temperatures of 20 and 30 °C, respectively. Oxidative stress (1 mM H2O2) and hemin limitation had no significant effect on mRNA levels. Determination of ialB protein amounts using SDS–PAGE and immunoblotting showed the greatest amounts of ialB under acidic conditions or at 20 °C. The least amount of ialB was synthesized under basic conditions or at 37 °C. The viability of wild-type B. bacilliformis under the various experimental culture conditions was determined and found not to affect ialB mRNA amounts in these experiments. Finally, we compared the survival of wild-type and ialB mutant B. bacilliformis and found no difference in the viability of these two strains, demonstrating that ialB does not aid bacterial survival under these conditions.
PMCID: PMC4104752  PMID: 12668141
Bartonella bacilliformis; Invasion-associated locus B (ialB) gene; Erythrocyte adherence and invasion; Bacterial pathogenesis
3.  Genetics of Coxiella burnetii: on the path of specialization 
Future microbiology  2011;6(11):1297-1314.
Coxiella burnetii is an extremely infectious, zoonotic agent that causes Q fever in humans. With the exception of New Zealand, the bacterium is distributed worldwide. Coxiella is classified as a select agent based on its past and potential use as a bioweapon and its threat to public health. Despite decades of research, we know relatively little regarding Coxiella’s molecular pathogenesis, and a vaccine is not widely available. This article briefly reviews the unusual genetics of C. burnetii; a pathogen that retains telltale genetic mementos collected over the course of its evolutionary path from a free-living bacterium to an obligate intracellular parasite of eukaryotic host cell phagosomes. Understanding why these genetic elements are maintained may help us better understand the biology of this fascinating pathogen.
doi:10.2217/fmb.11.116
PMCID: PMC4104754  PMID: 22082290
chromosome; Coxiella; genome reduction; genomics; genotype; plasmids; Q fever; selfish genetic elements
4.  Oroya Fever and Verruga Peruana: Bartonelloses Unique to South America 
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.
doi:10.1371/journal.pntd.0002919
PMCID: PMC4102455  PMID: 25032975
5.  Identification of Novel Small RNAs and Characterization of the 6S RNA of Coxiella burnetii 
PLoS ONE  2014;9(6):e100147.
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.
doi:10.1371/journal.pone.0100147
PMCID: PMC4064990  PMID: 24949863
6.  Kinetic models reveal the in vivo mechanisms of mutagenesis in microbes and man 
Mutation research  2012;752(2):129-137.
This review summarizes the evidence indicating that mutagenic mechanisms in vivo are essentially the same in all living cells. Unique metabolic reactions to a particular environmental stress apparently target specific genes for increased rates of transcription and mutation, resulting in higher mutation rates for those genes most likely to solve the problem. Kinetic models which have demonstrated predictive value are described and are shown to simulate mutagenesis in vivo in E. coli, the p53 tumor suppressor gene, and somatic hypermutation. In all three models, direct correlations are seen between mutation frequencies and transcription rates. G and C nucleosides in single-stranded DNA (ssDNA) are intrinsically mutable, and G and C silent mutations in p53 and in VH framework regions provide compelling evidence for intrinsic mechanisms of mutability, since mutation outcomes are neutral and are not selected. During transcription, the availability of unpaired bases in the ssDNA of secondary structures is rate-limiting for, and determines the frequency of mutations in vivo. In vitro analyses also verify the conclusion that intrinsically mutable bases are in fact located in ssDNA loops of predicted secondary structures.
doi:10.1016/j.mrrev.2012.12.003
PMCID: PMC3631585  PMID: 23274173
mutagenesis; E. coli; p53; somatic hypermutation; kinetic models
7.  Evolution of coordinated mutagenesis and somatic hypermutation in VH5 
Molecular immunology  2011;49(3):537-548.
The VH5 human antibody gene was analyzed using a computer program (mfg) which simulates transcription, to better understand transcription-driven mutagenesis events that occur during “phase 1” of somatic hypermutation. Results show that the great majority of mutations in the non-transcribed strand occur within loops of two predicted high-stability stem-loop structures, termed SLS 14.9 and SLS 13.9. In fact, 89% of the 2,505 mutations reported are within the encoded complementarity-determining region (CDR) and occur in loops of these high-stability structures. In vitro studies were also done and verified the existence of SLS 14.9. Following the formation of SLS 14.9 and SLS 13.9, a sustained period of transcriptional activity occurs within a window size of 60-70 nucleotides. During this period, the stability of these two SLSs does not change, and may provide the substrate for base exchanges and mutagenesis. The data suggest that many mutable bases are exposed simultaneously at pause sites, allowing for coordinated mutagenesis.
doi:10.1016/j.molimm.2011.10.001
PMCID: PMC3235918  PMID: 22056943
8.  The roles of transcription and genotoxins underlying p53 mutagenesis in vivo 
Carcinogenesis  2011;32(10):1559-1567.
Transcription drives supercoiling which forms and stabilizes single-stranded (ss) DNA secondary structures with loops exposing G and C bases that are intrinsically mutable and vulnerable to non-enzymatic hydrolytic reactions. Since many studies in prokaryotes have shown direct correlations between the frequencies of transcription and mutation, we conducted in silico analyses using the computer program, mfg, which simulates transcription and predicts the location of known mutable bases in loops of high-stability secondary structures. Mfg analyses of the p53 tumor suppressor gene predicted the location of mutable bases and mutation frequencies correlated with the extent to which these mutable bases were exposed in secondary structures. In vitro analyses have now confirmed that the 12 most mutable bases in p53 are in fact located in predicted ssDNA loops of these structures. Data show that genotoxins have two independent effects on mutagenesis and the incidence of cancer: Firstly, they activate p53 transcription, which increases the number of exposed mutable bases and also increases mutation frequency. Secondly, genotoxins increase the frequency of G-to-T transversions resulting in a decrease in G-to-A and C mutations. This precise compensatory shift in the ‘fate’ of G mutations has no impact on mutation frequency. Moreover, it is consistent with our proposed mechanism of mutagenesis in which the frequency of G exposure in ssDNA via transcription is rate limiting for mutation frequency in vivo.
doi:10.1093/carcin/bgr177
PMCID: PMC3179427  PMID: 21803733
9.  A unique Coxiella burnetii lipoprotein involved in metal binding (LimB) 
Microbiology  2011;157(Pt 4):966-976.
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.
doi:10.1099/mic.0.046649-0
PMCID: PMC3139440  PMID: 21212117
10.  Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii▿ 
Journal of Bacteriology  2011;193(19):5292-5299.
The 23S rRNA gene of Coxiella burnetii, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in Coxiella's biology, we determined the intrinsic stability time periods (in vitro half-lives) of the encoded ribozymes to be ∼15 days for L1917 and ∼5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA. In vivo half-lives for both introns were also determined to be ∼11 min by the use of RNase protection assays and an Escherichia coli model. Intron RNAs were quantified in synchronous cultures of C. burnetii and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the Coxiella intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The in vitro efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant Coxiella RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator in vivo.
doi:10.1128/JB.05472-11
PMCID: PMC3187410  PMID: 21803999
11.  Pentamidine inhibits Coxiella burnetii growth and 23S rRNA intron splicing in vitro 
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.
doi:10.1016/j.ijantimicag.2010.05.017
PMCID: PMC2926257  PMID: 20599360
Coxiella; Pentamidine; Group I intron; RNA splicing
12.  A DNA-Binding Peroxiredoxin of Coxiella burnetii Is Involved in Countering Oxidative Stress during Exponential-Phase Growth▿  
Journal of Bacteriology  2010;192(8):2077-2084.
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.
doi:10.1128/JB.01324-09
PMCID: PMC2849434  PMID: 20173000
13.  Pestilence, persistence and pathogenicity: infection strategies of Bartonella 
Future microbiology  2009;4:743-758.
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.
doi:10.2217/fmb.09.41
PMCID: PMC2754412  PMID: 19659429
angiogenesis; Bartonella; bartonellosis; hemotrophy; infection; virulence
15.  A Unique Group I Intron in Coxiella burnetii Is a Natural Splice Mutant▿  
Journal of Bacteriology  2009;191(12):4044-4046.
Cbu.L1917, a group I intron present in the 23S rRNA gene of Coxiella burnetii, possesses a unique 3′-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro.
doi:10.1128/JB.00359-09
PMCID: PMC2698393  PMID: 19376857
16.  VH gene transcription creates stabilized secondary structures for coordinated mutagenesis during somatic hypermutation 
Molecular immunology  2008;45(13):3589-3599.
During the adaptive immune response, antigen challenge triggers a million-fold increase in mutation rates in the variable region antibody genes. The frequency of mutation is causally and directly linked to transcription, which provides ssDNA and drives supercoiling that stabilizes secondary structures containing unpaired, intrinsically mutable bases. Simulation analysis of transcription in VH5 reveals a dominant 65 nt secondary structure in the non-transcribed strand containing six sites of mutable ssDNA that have also been identified independently in human B cell lines and in primary mouse B cells. This dominant structure inter-converts briefly with less stable structures and is formed repeatedly during transcription, due to periodic pauses and backtracking. In effect, this creates a stable yet dynamic “mutability platform” consisting of ever-changing patterns of unpaired bases that are simultaneously exposed and therefore able to coordinate mutagenesis. Such a complex of secondary structures may be the source of ssDNA for enzyme-based diversification, which ultimately results in high affinity antibodies.
doi:10.1016/j.molimm.2008.02.030
PMCID: PMC2570340  PMID: 18585784
Somatic hypermutation; Transcription-directed mutagenesis; Secondary structures; Supercoiling
17.  II. Correlations between secondary structure stability and mutation frequency during somatic hypermutation 
Molecular immunology  2008;45(13):3600-3608.
The role of secondary structures and base mutability at different levels of transcription and supercoiling is analyzed in variable region antibody genes VH5, VH94 and VH186.2. The data are consistent with a model of somatic hypermutation in which increasing levels of transcription and secondary structure stability correlate with the initial formation of successive mutable sites. Encoded differences exist in stem length and the number of GC pairs at low versus high levels of transcription in CDRs. These circumstances simplify the complexities of coordinating mutagenesis by confining this process to each mutable site successively, as they form in response to increasing levels of transcription during affinity maturation.
doi:10.1016/j.molimm.2008.05.012
PMCID: PMC2529022  PMID: 18584870
18.  Function, Regulation, and Transcriptional Organization of the Hemin Utilization Locus of Bartonella quintana▿ †  
Infection and Immunity  2008;77(1):307-316.
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.
doi:10.1128/IAI.01194-08
PMCID: PMC2612243  PMID: 18981245
19.  Toxic Introns and Parasitic Intein in Coxiella burnetii: Legacies of a Promiscuous Past▿  
Journal of Bacteriology  2008;190(17):5934-5943.
The genome of the obligate intracellular pathogen Coxiella burnetii contains a large number of selfish genetic elements, including two group I introns (Cbu.L1917 and Cbu.L1951) and an intervening sequence that interrupts the 23S rRNA gene, an intein (Cbu.DnaB) within dnaB and 29 insertion sequences. Here, we describe the ability of the intron-encoded RNAs (ribozymes) to retard bacterial growth rate (toxicity) and examine the functionality and phylogenetic history of Cbu.DnaB. When expressed in Escherichia coli, both introns repressed growth, with Cbu.L1917 being more inhibitory. Both ribozymes were found to associate with ribosomes of Coxiella and E. coli. In addition, ribozymes significantly reduced in vitro luciferase translation, again with Cbu.L1917 being more inhibitory. We analyzed the relative quantities of ribozymes and genomes throughout a 14-day growth cycle of C. burnetii and found that they were inversely correlated, suggesting that the ribozymes have a negative effect on Coxiella's growth. We determined possible sites for ribozyme associations with 23S rRNA that could explain the observed toxicities. Further research is needed to determine whether the introns are being positively selected because they promote bacterial persistence or whether they were fixed in the population due to genetic drift. The intein, Cbu.DnaB, is able to self-splice, leaving the host protein intact and presumably functional. Similar inteins have been found in two extremophilic bacteria (Alkalilimnicola ehrlichei and Halorhodospira halophila) that are distantly related to Coxiella, making it difficult to determine whether the intein was acquired by horizontal gene transfer or was vertically inherited from a common ancestor.
doi:10.1128/JB.00602-08
PMCID: PMC2519523  PMID: 18606739
20.  Bartonella bacilliformis GroEL; Effect on Growth of Human Vascular Endothelial Cells in Infected Co-Cultures 
Bartonella are the only bacteria known to induce angioproliferative lesions of the human vasculature and liver during infection. Previous work from our lab suggests that GroEL participates in the mitogenic response observed in HUVEC cultures supplemented with the soluble fraction of Bartonella bacilliformis. Work in this study shows that exposure to high concentrations of the fraction is actually cytotoxic for HUVECs. To analyze this phenomenon, live B. bacillformis - HUVEC co-cultures were employed to study the effect of excess bacterial GroEL on the host cell during active infection. Four B. bacilliformis strains were generated to produce varying levels of GroEL. HUVEC co-cultures with LSS100, a strain that synthesizes markedly greater quantities of GroEL relative to others, significantly accelerates apoptosis of the co-cultured HUVECs relative to other strains. Acceleration of apoptosis can be inhibited by Z-VAD-FMK, a pan-caspase inhibitor. Time course data show that at 18 h of infection, both LSS100 and control strains significantly inhibit spontaneous apoptosis of co-cultured HUVECs, as previously reported for other Bartonella species. However, by 48 h LSS100 significantly increases apoptosis of the host cell. We hypothesize that intracellular Bartonella GroEL functions as an HSP60 analog, a eukaryotic orthologue known to accelerate procaspase 3 activation by enhancing its vulnerability to upstream activator caspases. These data suggest another strategy whereby Bartonella may regulate host cell growth.
doi:10.1196/annals.1355.046
PMCID: PMC1817666  PMID: 16481529
21.  The Unusual 23S rRNA Gene of Coxiella burnetii: Two Self-Splicing Group I Introns Flank a 34-Base-Pair Exon, and One Element Lacks the Canonical ΩG▿  
Journal of Bacteriology  2007;189(18):6572-6579.
We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed ΩG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (≥99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer.
doi:10.1128/JB.00812-07
PMCID: PMC2045182  PMID: 17644584
22.  Transcriptional Regulation of the Heme Binding Protein Gene Family of Bartonella quintana Is Accomplished by a Novel Promoter Element and Iron Response Regulator▿ †  
Infection and Immunity  2007;75(9):4373-4385.
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.
doi:10.1128/IAI.00497-07
PMCID: PMC1951173  PMID: 17576755
23.  Proteomic and Immunoblot Analyses of Bartonella quintana Total Membrane Proteins Identify Antigens Recognized by Sera from Infected Patients▿  
Infection and Immunity  2007;75(5):2548-2561.
Bartonella quintana is a fastidious, gram-negative, rod-shaped bacterium that causes prolonged bacteremia in immunocompetent humans and severe infections in immunocompromised individuals. We sought to define the outer membrane subproteome of B. quintana in order to obtain insight into the biology and pathogenesis of this emerging pathogen and to identify the predominant B. quintana antigens targeted by the human immune system during infection. We isolated the total membrane proteins of B. quintana and identified 60 proteins by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass fingerprinting. Using the newly constructed proteome map, we then utilized two-dimensional immunoblotting with sera from 21 B. quintana-infected patients to identify 24 consistently recognized, immunoreactive B. quintana antigens that have potential relevance for pathogenesis and diagnosis. Among the outer membrane proteins, the variably expressed outer membrane protein adhesins (VompA and VompB), peptidyl-prolyl cis-trans-isomerase (PpI), and hemin-binding protein E (HbpE) were recognized most frequently by sera from patients, which is consistent with surface expression of these virulence factors during human infection.
doi:10.1128/IAI.01974-06
PMCID: PMC1865797  PMID: 17307937
24.  Characterization and expression analysis of the groESL operon of Bartonella bacilliformis 
Gene  2005;359:53-62.
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.
doi:10.1016/j.gene.2005.06.031
PMCID: PMC1885459  PMID: 16126349
GroESL operon; Bartonella; Heat shock protein; Chaperonin; GroEL; GroES
25.  Environmental Signals Generate a Differential and Coordinated Expression of the Heme Receptor Gene Family of Bartonella quintana  
Infection and Immunity  2006;74(6):3251-3261.
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.
doi:10.1128/IAI.00245-06
PMCID: PMC1479232  PMID: 16714552

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