Nicole C. Ammerman

Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

Background The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. Methodology/Principal Findings Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFδ, is an artifact of the original genome assembly. Conclusion/Significance Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree.

Targeting DnaN for tuberculosis therapy using novel griselimycins

New for old—TB drug development Tuberculosis (TB) is a global health threat for which there is only lengthy drug treatment. Patients need to consume multiple tablets over several months and frequently fail to complete their treatment. Consequently, drug-resistant strains of the pathogen have emerged, which add to the threat. Kling et al. revisited a natural product called griselimycin, extracted from the same organism that produced the prototype anti-TB drug, streptomycin. Unmodified griselimycin has poor pharmacological properties. However, one synthetic derivative had improved oral uptake and penetrated cells of the immune system that harbor the TB mycobacterium. In combination with other drugs, the griselimycin derivative showed high potency in mice with TB. Science, this issue p. 1106 A griselimycin-derived drug that blocks the DNA polymerase sliding clamp is a potent anti-tuberculosis lead. The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.

Acceleration of Tuberculosis Treatment by Adjunctive Therapy with Verapamil as an Efflux Inhibitor

RATIONALE A major priority in tuberculosis (TB) is to reduce effective treatment times and emergence of resistance. Recent studies in macrophages and zebrafish show that inhibition of mycobacterial efflux pumps with verapamil reduces the bacterial drug tolerance and may enhance drug efficacy. OBJECTIVES Using mice, a mammalian model known to predict human treatment responses, and selecting conservative human bioequivalent doses, we tested verapamil as an adjunctive drug together with standard TB chemotherapy. As verapamil is a substrate for CYP3A4, which is induced by rifampin, we evaluated the pharmacokinetic/pharmacodynamic relationships of verapamil and rifampin coadministration in mice. METHODS Using doses that achieve human bioequivalent levels matched to those of standard verapamil, but lower than those of extended release verapamil, we evaluated the activity of verapamil added to standard chemotherapy in both C3HeB/FeJ (which produce necrotic granulomas) and the wild-type background C3H/HeJ mouse strains. Relapse rates were assessed after 16, 20, and 24 weeks of treatment in mice. MEASUREMENTS AND MAIN RESULTS We determined that a dose adjustment of verapamil by 1.5-fold is required to compensate for concurrent use of rifampin during TB treatment. We found that standard TB chemotherapy plus verapamil accelerates bacterial clearance in C3HeB/FeJ mice with near sterilization, and significantly lowers relapse rates in just 4 months of treatment when compared with mice receiving standard therapy alone. CONCLUSIONS These data demonstrate treatment shortening by verapamil adjunctive therapy in mice, and strongly support further study of verapamil and other efflux pump inhibitors in human TB.

An Anomalous Type IV Secretion System in Rickettsia Is Evolutionarily Conserved

Background Bacterial type IV secretion systems (T4SSs) comprise a diverse transporter family functioning in conjugation, competence, and effector molecule (DNA and/or protein) translocation. Thirteen genome sequences from Rickettsia, obligate intracellular symbionts/pathogens of a wide range of eukaryotes, have revealed a reduced T4SS relative to the Agrobacterium tumefaciens archetype (vir). However, the Rickettsia T4SS has not been functionally characterized for its role in symbiosis/virulence, and none of its substrates are known. Results Superimposition of T4SS structural/functional information over previously identified Rickettsia components implicate a functional Rickettsia T4SS. virB4, virB8 and virB9 are duplicated, yet only one copy of each has the conserved features of similar genes in other T4SSs. An extraordinarily duplicated VirB6 gene encodes five hydrophobic proteins conserved only in a short region known to be involved in DNA transfer in A. tumefaciens. virB1, virB2 and virB7 are newly identified, revealing a Rickettsia T4SS lacking only virB5 relative to the vir archetype. Phylogeny estimation suggests vertical inheritance of all components, despite gene rearrangements into an archipelago of five islets. Similarities of Rickettsia VirB7/VirB9 to ComB7/ComB9 proteins of ε-proteobacteria, as well as phylogenetic affinities to the Legionella lvh T4SS, imply the Rickettsiales ancestor acquired a vir-like locus from distantly related bacteria, perhaps while residing in a protozoan host. Modern modifications of these systems likely reflect diversification with various eukaryotic host cells. Conclusion We present the rvh (Rickettsiales vir homolog) T4SS, an evolutionary conserved transporter with an unknown role in rickettsial biology. This work lays the foundation for future laboratory characterization of this system, and also identifies the Legionella lvh T4SS as a suitable genetic model.

Indoleamides are active against drug-resistant Mycobacterium tuberculosis

Responsible for nearly two million deaths each year, the infectious disease tuberculosis remains a serious global health challenge. The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis confounds control efforts, and new drugs with novel molecular targets are desperately needed. Here we describe lead compounds, the indoleamides, with potent activity against both drug-susceptible and drug-resistant strains of M. tuberculosis by targeting the mycolic acid transporter MmpL3. We identify a single mutation in mmpL3 which confers high resistance to the indoleamide class while remaining susceptible to currently used first- and second-line tuberculosis drugs, indicating a lack of cross-resistance. Importantly, an indoleamide derivative exhibits dose-dependent anti-mycobacterial activity when orally administered to M. tuberculosis-infected mice. The bioavailability of the indoleamides, combined with their ability to kill tubercle bacilli, indicates great potential for translational developments of this structure class for the treatment of drug-resistant tuberculosis.

Assessment of Clofazimine Activity in a Second-Line Regimen for Tuberculosis in Mice

RATIONALE Although observational studies suggest that clofazimine-containing regimens are highly active against drug-resistant tuberculosis, the contribution of clofazimine for the treatment of this disease has never been systematically evaluated. OBJECTIVES Our goal was to directly compare the activity of a standard second-line drug regimen with or without the addition of clofazimine in a mouse model of multidrug-resistant tuberculosis. Our comparative outcomes included time to culture conversion in the mouse lungs and the percentage of relapses after treatment cessation. METHODS Mice were aerosol-infected with an isoniazid-resistant (as a surrogate of multidrug-resistant) strain of Mycobacterium tuberculosis. Treatment, which was administered for 5 to 9 months, was initiated 2 weeks after infection and comprised the following second-line regimen: daily (5 d/wk) moxifloxacin, ethambutol, and pyrazinamide, supplemented with amikacin during the first 2 months. One-half of the mice also received daily clofazimine. The decline in lung bacterial load was assessed monthly using charcoal-containing agar to reduce clofazimine carryover. Relapse was assessed 6 months after treatment cessation. MEASUREMENTS AND MAIN RESULTS After 2 months, the bacillary load in lungs was reduced from 9.74 log10 at baseline to 3.61 and 4.68 in mice treated with or without clofazimine, respectively (P < 0.001). Mice treated with clofazimine were culture-negative after 5 months, whereas all mice treated without clofazimine remained heavily culture-positive for the entire 9 months of the study. The relapse rate was 7% among mice treated with clofazimine for 8 to 9 months. CONCLUSIONS The clofazimine contribution was substantial in these experimental conditions.

Clofazimine shortens the duration of the first-line treatment regimen for experimental chemotherapy of tuberculosis

Significance The infectious disease tuberculosis (TB) is a major public health problem that affects millions of people worldwide. TB treatment consists of a multidrug regimen that needs to be taken for a minimum of 6 mo, and lack of adherence to this regimen is associated with treatment failure and emergence of drug resistance. In a mouse model of TB chemotherapy, we have found that inclusion of the antileprosy drug clofazimine in the first-line regimen for TB reduces the duration of treatment necessary to achieve relapse-free cure from 6 mo to 3 mo. Our data suggest that clofazimine, a drug already known to be safe for long-term administration to patients with leprosy, has the potential to significantly shorten the duration of TB treatment. A key drug for the treatment of leprosy, clofazimine has recently been associated with highly effective and significantly shortened regimens for the treatment of multidrug-resistant tuberculosis (TB). Consequently, we hypothesized that clofazimine may also shorten the duration of treatment for drug-susceptible TB. We conducted a controlled trial in the mouse model of TB chemotherapy comparing the activity of the 6-mo standard regimen for TB treatment, i.e., 2 mo of daily rifampin, isoniazid, pyrazinamide, and ethambutol followed by 4 mo of rifampin and isoniazid, with a 4-mo clofazimine-containing regimen: 2 mo of daily rifampin, isoniazid, pyrazinamide, and clofazimine followed by 2 mo of rifampin, isoniazid, and clofazimine. Treatment efficacy was assessed on the basis of Mycobacterium tuberculosis colony counts in the lungs and spleens during treatment and on the proportion of mice with culture-positive relapse 6 mo after treatment cessation. No additive effect of clofazimine was observed after the first week of treatment, but, by the second week of treatment, the colony counts were significantly lower in the clofazimine-treated mice than in the mice receiving the standard regimen. Lung culture conversion was obtained after 3 and 5 mo in mice treated with the clofazimine-containing and standard regimens, respectively, and relapse-free cure was obtained after 3 and 6 mo of treatment with the clofazimine-containing and standard regimens, respectively. Thus, clofazimine is a promising anti-TB drug with the potential to shorten the duration of TB chemotherapy by at least half (3 mo vs. 6 mo) in the mouse model of TB.

Preliminary Structure-Activity Relationships and Biological Evaluation of Novel Antitubercular Indolecarboxamide Derivatives Against Drug-Susceptible and Drug-Resistant Mycobacterium tuberculosis Strains

Tuberculosis (TB) remains one of the leading causes of mortality and morbidity worldwide, with approximately one-third of the worlds population infected with latent TB. This is further aggravated by HIV coinfection and the emergence of multidrug- and extensively drug-resistant (MDR and XDR, respectively) TB; hence the quest for highly effective antitubercular drugs with novel modes of action is imperative. We report herein the discovery of an indole-2-carboxamide analogue, 3, as a highly potent antitubercular agent, and the subsequent chemical modifications aimed at establishing a preliminary body of structure-activity relationships (SARs). These efforts led to the identification of three molecules (12-14) possessing an exceptional activity in the low nanomolar range against actively replicating Mycobacterium tuberculosis , with minimum inhibitory concentration (MIC) values lower than those of the most prominent antitubercular agents currently in use. These compounds were also devoid of apparent toxicity to Vero cells. Importantly, compound 12 was found to be active against the tested XDR-TB strains and orally active in the serum inhibition titration assay.

Spotted-Fever Group Rickettsia in Dermacentor variabilis, Maryland

Three-hundred ninety-two adult Dermacentor variabilis were collected from six Maryland counties during the spring, summer, and fall of 2002. Infection prevalence for spotted fever group Rickettsia was 3.8%, as determined by polymerase chain reaction. Single strand conformational polymorphism (SSCP) analysis followed by sequencing indicated that all infections represented a single rickettsial taxon, Rickettsia montanensis.

Growth and Maintenance of Vero Cell Lines

Vero cells are derived from the kidney of an African green monkey, and are one of the more commonly used mammalian continuous cell lines in microbiology and molecular and cell biology research. This unit includes protocols for the growth and maintenance of Vero cell lines in a research laboratory setting. Curr. Protoc. Microbiol. 11:A.4E.1‐A.4E.7.