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The mechanism of action (MoA) of a clickable fatty acid analogue 8-(2-cyclobuten-1-yl)octanoic acid (DA-CB) has been investigated for the first time. Proteomics, metabolomics, and lipidomics were combined with a network analysis to investigate the MoA of DA-CB against

Graphical abstract, Tuberculosis (TB), one of the oldest and deadliest bacterial diseases, continues to cause serious global economic, health, and social problems. Current TB treatments are lengthy, expensive, and routinely ineffective against emerging drug resistant strains. Thus, there is an urgent need for the identification and development of novel TB drugs possessing comprehensive and specific mechanisms of action (MoAs). Metabolomics is a valuable approach to elucidating the MoA, toxicity, and potency of promising chemical leads, which is a critical step of the drug discovery process. Recent advances in metabolomics methodologies for deciphering MoAs include high-throughput screening techniques, the integration of multiple omics methods, mass spectrometry imaging, and software for automated analysis. This review describes recently introduced metabolomics methodologies and techniques for drug discovery, highlighting specific applications to the discovery of new antitubercular drugs and the elucidation of their MoAs.

Native hosts for the bacterial agent that causes Johne's disease are ruminants, which include cattle, sheep and goats among others. These large animals are often too costly to be used in testing experimental vaccines. In this chapter, we provide detailed methods to use an inexpensive and more manageable animal host, the ferret, to test efficacy and immunogenicity of live-attenuated Mycobacterium avium subspecies paratuberculosis (MAP) mutant strains prior to consideration as vaccine candidates.

Babesiosis is a disease caused by tickborne hemoprotozoan apicomplexan parasites of the genus Babesia that negatively impacts public health and food security worldwide. Development of effective and sustainable vaccines against babesiosis is currently hindered in part by the absence of definitive host correlates of protection. Despite that, studies in Babesia microti and Babesia bovis, major causative agents of human and bovine babesiosis, respectively, suggest that early activation of innate immune responses is crucial for vertebrates to survive acute infection. Trained immunity (TI) is defined as the development of memory in vertebrate innate immune cells, allowing more efficient responses to subsequent specific and non-specific challenges. Considering that Mycobacterium bovis bacillus Calmette-Guerin (BCG), a widely used anti-tuberculosis attenuated vaccine, induces strong TI pro-inflammatory responses, we hypothesize that BCG TI may protect vertebrates against acute babesiosis. This premise is supported by early investigations demonstrating that BCG inoculation protects mice against experimental B. microti infection and recent observations that BCG vaccination decreases the severity of malaria in children infected with Plasmodium falciparum, a Babesia-related parasite. We also discuss the potential use of TI in conjunction with recombinant BCG vaccines expressing Babesia immunogens. In conclusion, by concentrating on human and bovine babesiosis, herein we intend to raise awareness of BCG TI as a strategy to efficiently control Babesia infection.

Intracellular iron concentration is tightly regulated to maintain cell viability. Iron plays important roles in electron transport, nucleic acid synthesis, and oxidative stress. A Mycobacterium avium subsp. paratuberculosis (MAP)-specific genomic island carries a putative metal transport operon that includes MAP3773c, which encodes a Fur-like protein. Although well characterized as a global regulator of iron homeostasis in multiple bacteria, the function of Fur (ferric uptake regulator) in MAP is unknown as this organism also carries IdeR (iron dependent regulator), a native iron regulatory protein specific to mycobacteria. Computational analysis using PRODORIC identified 23 different pathways involved in respiration, metabolism, and virulence that were likely regulated by MAP3773c. Thus, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was performed to confirm the putative regulon of MAP3773c (Fur-like protein) in MAP. ChIP-Seq revealed enriched binding to 58 regions by Fur under iron-replete and -deplete conditions, located mostly within open reading frames (ORFs). Three ChIP peaks were identified in genes that are directly related to iron regulation: MAP3638c (hemophore-like protein), MAP3736c (Fur box), and MAP3776c (ABC transporter). Fur box consensus sequence was identified, and binding specificity and dependence on Mn2+ availability was confirmed by a chemiluminescent electrophoresis mobility shift assay (EMSA). The results confirmed that MAP3773c is a Fur ortholog that recognizes a 19 bp DNA sequence motif (Fur box) and it is involved in metal homeostasis. This work provides a regulatory network of MAP Fur binding sites during iron-replete and -deplete conditions, highlighting unique properties of Fur regulon in MAP.

In mycobacteria, D-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form D-alanine is through the racemization of L-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of D-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild type mc(2)155 in the absence of D-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc(2)155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented D-alanine. In the absence of D-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential D-alanine required for survival. The process is reversed when D-alanine is available, in which the D-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis, and that specific Alr inhibitors will have no bactericidal action.

This article reviews the discovery, structure, secretion, and role in microbial evolution and immuno-pathogenesis of a group of Mycobacterium tuberculosis acidic glycine rich proteins containing multiple copies of repetitive sequences. The classification of these into PE and PPE proteins is based on the presence of proline-glutamic acid (PE) or proline-proline-glutamic acid (PPE) residues at their N-terminal domains. The structural basis for their interactions in PE-PPE pairs and with mycobacterial protein family secretion systems (ESX or Type VII) is described. We also illustrate the role of these proteins in mycobacterial evolution and immuno-pathogenesis. The emergence of the various lineages and subfamilies of PE and PPE proteins and their ancestral counterparts are described in the context of the interactions of the pathogen with host cells and the elicitation of humoral and cellular immune responses.

While transposon mutagenesis has been developed for Mycobacterium avium subspecies paratuberculosis (Map), relatively few laboratories have adopted this important genetic tool to examine gene function and essentiality. Here we describe the construction of a Map transposon library using the Himar1 mariner transposon, but concepts can also be applied to the Tn5367 transposon, which has also been used by our group. Delivery of the transposon is by a temperature-sensitive phagemid, ϕMycoMarT7, and plating transductants requires patience and specialized media due to length of incubation required to observe colonies. Several transposon mutants obtained from these libraries have been tested in vaccine and pathogenesis studies. By providing the following detailed protocol herein, we expect to demystify the procedure and encourage additional investigators to incorporate transposon mutagenesis in their studies on Johne's disease.

Mycobacterium avium subsp. paratuberculosis (MAP), the aetiological agent of Johne's disease, is one of the most important bacterial pathogens in ruminants. A thorough understanding of MAP pathogenesis is needed to develop new vaccines and diagnostic tests. The generation of comprehensive random transposon mutant libraries is a fundamental genetic technology to determine the role of genes in physiology and pathogenesis. In this study, whole MAP genome analysis compared the insertion sites for the mycobacterial transposon Tn5367 derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposon MycoMarT7 carrying the Himar1 transposase. We determined that only MycoMarT7 provides a random representation of insertions in 99 % of all MAP genes. Analysis of the MAP K-10 genome indicated that 710 of all ORFs do not possess IS1096 recognition sites, while only 37 do not have the recognition site for MycoMarT7. Thus, a significant number of MAP genes remain underrepresented in insertion libraries from IS1096-derived transposons. Analysis of MycoMarT7 and Tn5367 mutants showed that Tn5367 has a predilection to insert within intergenic regions, suggesting that MycoMarT7 is the more adequate for generating a comprehensive library. However, we uncovered the novel finding that both transposons have loci-dependent biases, with Tn5367 being the most skewed. These loci-dependent transposition biases led to an underestimation of the number of independent mutants required to generate a comprehensive mutant library, leading to an overestimation of essential genes. Herein, we also demonstrated a useful platform for gene discovery and analysis by isolating three novel mutants for each transposon.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in ruminants causing chronic diarrhea, malnutrition, and muscular wasting. Neonates and young animals are infected primarily by the fecal-oral route. MAP attaches to, translocates via the intestinal mucosa, and is phagocytosed by macrophages. The ensuing host cellular immune response leads to granulomatous enteritis characterized by a thick and corrugated intestinal wall. We review various tissue culture systems, ileal loops, and mice, goats, and cattle used to study MAP pathogenesis. MAP can be detected in clinical samples by microscopy, culturing, PCR, and an enzyme-linked immunosorbent assay. There are commercial vaccines that reduce clinical disease and shedding, unfortunately, their efficacies are limited and may not engender long-term protective immunity. Moreover, the potential linkage with Crohn's disease and other human diseases makes MAP a concern as a zoonotic pathogen. Potential therapies with anti-mycobacterial agents are also discussed. The completion of the MAP K-10 genome sequence has greatly improved our understanding of MAP pathogenesis. The analysis of this sequence has identified a wide range of gene functions involved in virulence, lipid metabolism, transcriptional regulation, and main metabolic pathways. We also review the transposons utilized to generate random transposon mutant libraries and the recent advances in the post-genomic era. This includes the generation and characterization of allelic exchange mutants, transcriptomic analysis, transposon mutant banks analysis, new efforts to generate comprehensive mutant libraries, and the application of transposon site hybridization mutagenesis and transposon sequencing for global analysis of the MAP genome. Further analysis of candidate vaccine strains development is also provided with critical discussions on their benefits and shortcomings, and strategies to develop a highly efficacious live-attenuated vaccine capable of differentiating infected from vaccinated animals.

d-Cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR) drug resistant strains of Mycobacterium tuberculosis . d-Cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of alanine racemase (Alr) and d-alanine-d-alanine ligase (Ddl). Although the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of d-alanyl-d-alanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of d-alanine, thus allowing DCS to outcompete d-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce d-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.

Background We recently reported the identification of Bacillus sp. NRRL B-14911 that induces heart autoimmunity by generating cardiac-reactive T cells through molecular mimicry. This marine bacterium was originally isolated from the Gulf of Mexico, but no associations with human diseases were reported. Therefore, to characterize its biological and medical significance, we sought to determine and analyze the complete genome sequence of Bacillus sp. NRRL B-14911. Results Based on the phylogenetic analysis of 16S ribosomal RNA (rRNA) genes, sequence analysis of the 16S-23S rDNA intergenic transcribed spacers, phenotypic microarray, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we propose that this organism belongs to the species Bacillus infantis, previously shown to be associated with sepsis in a newborn child. Analysis of the complete genome of Bacillus sp. NRRL B-14911 revealed several virulence factors including adhesins, invasins, colonization factors, siderophores and transporters. Likewise, the bacterial genome encodes a wide range of methyl transferases, transporters, enzymatic and biochemical pathways, and insertion sequence elements that are distinct from other closely related bacilli. Conclusions The complete genome sequence of Bacillus sp. NRRL B-14911 provided in this study may facilitate genetic manipulations to assess gene functions associated with bacterial survival and virulence. Additionally, this bacterium may serve as a useful tool to establish a disease model that permits systematic analysis of autoimmune events in various susceptible rodent strains. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2900-2) contains supplementary material, which is available to authorized users.

New strategies are needed to circumvent increasing outbreaks of resistant strains of pathogens and to expand the dwindling supply of effective antimicrobials. A common impediment to drug development is the lack of an easy approach to determine the in vivo mechanism of action and efficacy of novel drug leads. Towards this end, we describe an unbiased approach to predict in vivo mechanisms of action from NMR metabolomics data. Mycobacterium smegmatis, a non-pathogenic model organism for Mycobacterium tuberculosis, was treated with 12 known drugs and 3 chemical leads identified from a cell-based assay. NMR analysis of drug-induced changes to the M. smegmatis metabolome resulted in distinct clustering patterns correlating with in vivo drug activity. The clustering of novel chemical leads relative to known drugs provides a mean to identify a protein target or predict in vivo activity.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein ( P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera ( P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle ( P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis -infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.

d -Alanine: d -alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two d -alanine ( d -Ala) molecules to form the d -alanyl: d -alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. d -Cycloserine (DCS), an analog of d -Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 Å. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoined by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and d -alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and d -Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for d -Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC 50 ) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic intestinal infection in ruminants. Adenosine 5′-Triphosphate (ATP) has been reported to induce killing of several Mycobacterium species in human and murine macrophages. We investigated whether ATP secreted from M. avium subsp. paratuberculosis -infected bovine monocytes affects intracellular survival of the bacilli. Bovine monocytes constitutively secreted ATP during an 8-day incubation period in vitro ; however, M. avium subsp. paratuberculosis infection did not enhance ATP release. Removal of extracellular ATP by the addition of apyrase increased the viability of infected monocytes, but surprisingly decreased the number of viable intracellular bacilli. In contrast to previous reports, addition of extracellular ATP (1 mM) increased intracellular survival of M. avium subsp. paratuberculosis in bovine monocytes. Neither apyrase nor ATP altered production of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by bovine monocytes. These results suggest that ATP release from infected bovine monocytes improves, rather than decreases, the intracellular survival of M. avium subsp. paratuberculosis .

We have recently described the development of a luminescentMycobacterium paratuberculosisstrain of bovine origin expressing theluxABgenes ofVibrio harveyi. With this luminescent isolate, fastidious and costly enumeration of CFU by plating them on agar can be replaced by easy and rapid luminometry. Here, we have reevaluated the effect ofSlc11a1(formerlyNramp1) polymorphism on susceptibility toM. paratuberculosis, using this luminometric method. A series of inbred mouse strains were infected intravenously with luminescentM. paratuberculosisS-23 and monitored for bacterial replication in spleen, liver, and lungs for 12 weeks. The results indicate that, as forMycobacterium aviumsubsp.avium, innate resistance to infection is genetically controlled bySlc11a1. In BALB/c, congenic BALB.B10-H2b(BALB/c background;H-2b), C57BL/6, and beige C57BL/6bg/bgmice (allSlc11a1s), bacterial numbers in spleen and liver remained unchanged during the first 4 weeks of infection, whereas in DBA/2 and congenic BALB/c.DBA/2 (C.D2) mice (bothSlc11a1r) and in (C57BL/6 × DBA/2)F1mice (Slc11a1s/r), the bacterial numbers had decreased more than 10-fold at 4 weeks postinfection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver. Whereas bacterial numbers in the liver gradually decreased more than 100-fold in C57BL/6 mice between week 4 and week 12, bacterial numbers were stable in livers from BALB/c and beige C57BL/6bg/bgmice during this period. Mycobacterium-specific gamma interferon responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice.

Mycobacterium avium subsp. paratuberculosis is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. This disease has been difficult to control because of the lack of an effective vaccine. To address this need, we adapted a specialized transduction system originally developed for M. tuberculosis and modified it to improve the efficiency of allelic exchange in order to generate site-directed mutations in preselected M. avium subsp. paratuberculosis genes. With our novel optimized method, the allelic exchange frequency was 78 to 100% and the transduction frequency was 1.1 × 10 −7 to 2.9 × 10 −7 . Three genes were selected for mutagenesis: pknG and relA , which are genes that are known to be important virulence factors in M. tuberculosis and M. bovis , and lsr2 , a gene regulating lipid biosynthesis and antibiotic resistance. Mutants were successfully generated with a virulent strain of M. avium subsp. paratuberculosis ( M. avium subsp. paratuberculosis K10) and with a recombinant K10 strain expressing the green fluorescent protein gene, gfp . The improved efficiency of disruption of selected genes in M. avium subsp. paratuberculosis should accelerate development of additional mutants for vaccine testing and functional studies.

D-Cycloserine (DCS) is only used with multidrug-resistant strains of tuberculosis because of serious side effects. DCS is known to inhibit cell wall biosynthesis, but the in vivo lethal target is still unknown. We have applied NMR-based metabolomics combined with principal component analysis to monitor the in vivo effect of DCS on Mycobacterium smegmatis. Our analysis suggests DCS functions by inhibiting multiple protein targets.

Mycobacterium aviumsubsp.paratuberculosisis the etiologic agent of Johne's disease, a chronic granulomatous enteritis in ruminants. ATP has been reported to induce cell death of macrophages and killing ofMycobacteriumspecies in human and murine macrophages. In this study we investigated the short-term effect of ATP on the viability ofM. aviumsubsp.paratuberculosis-infected bovine mononuclear phagocytes and the bacilli within them. Addition of 5 mM ATP toM. aviumsubsp.paratuberculosis-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of 2′(3′)-O-(4-benzoylbenzoyl) ATP triethylammonium salt (Bz-ATP), which is a longer-lived ATP homologue and purinergic receptor agonist, significantly increased the uptake of YO-PRO, which is a marker for membrane pore activation by P2X receptors. Addition of Bz-ATP also stimulated lactate dehydrogenase release and caspase-3 activity in infected bovine monocytes. Neither ATP nor Bz-ATP reduced the survival ofM. aviumsubsp.paratuberculosisin bovine mononuclear phagocytes. Likewise, addition of ATP or Bz-ATP was cytotoxic to murine macrophage cell lines (RAW 264.7 and J774A.1 cells) but did not affect the intracellular survival ofM. aviumsubsp.paratuberculosis, nor were the numbers of viableMycobacterium aviumsubsp.aviumorMycobacterium bovisBCG cells altered in bovine mononuclear phagocytes or J774A.1 cells following ATP or Bz-ATP treatment. These data suggest that extracellular ATP does not induce the killing of intracellularM. aviumsubsp.paratuberculosisin bovine mononuclear phagocytes.

El conocimiento derivado del genoma de Mycobacterium tuberculosis, junto con el desarrollo de sofisticados sistemas para la manipulación genética del bacilo, ofrece la mayor promesa para el desarrollo de herramientas nuevas y más eficientes para prevenir y controlar la tuberculosis. Se han desarrollado métodos más eficientes para la inactivación de genes micobacterianos que se han convertido en el pilar de la genómica funcional micobacteriana. La generación de mutantes mediante la inactivación génica, apoyada directa o indirectamente por el desciframiento del genoma micobacteriano, ha permitido la generación de un número significativo de mutantes de M. tuberculosis. En algunos casos, el análisis de estas mutantes ha establecido relaciones entre los productos génicos y sus funciones en la fisiología y la patogenicidad de la micobacteria. En esta revisión se describen los estudios más representativos basados en dichas mutantes. Gene inactivation in Mycobacterium tuberculosis and its use in tuberculosis control and prevention Availability of the M. tuberculosis genome sequence and the development of sophisticated systems for genetic manipulation of bacilli offer the potential for new and effective tools to prevent and control tuberculosis. Efficient methods to inactivate mycobacterial genes have been developed. These methods have become the cornerstone for the application and development of mycobacterial functional genomics. Specific mutants are generated to establish the role of targetted genes associated with mycobacterial physiology and pathogenesis. Gene inactivation, supported directly or indirectly by the deciphering of the mycobacterial genome, has permitted the generation of large numbers of M. tuberculosis mutants. Analysis of these mutants has (in some cases) established relationships between gene products and their role in mycobacterial physiology and pathogenesis.

d- Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes d- alanine racemase (Alr) and d- alanine: d- alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis . We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of d- alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of l- and d- alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of l- over d- alanine. In all strains, DCS treatment led to a significant accumulation of l- alanine and a concomitant decease of d- alanine, with approximately a 20-fold excess of l- alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the d- alanine branch of mycobacterial peptidoglycan biosynthesis.

Mycobacterium avium causes disseminated infection in patients with acquired immune deficiency syndrome. Mycobacterium tuberculosis is a pathogen associated with the deaths of millions of people worldwide annually. Effective therapeutic regimens exist that are limited by the emergence of drug resistance and the inability of antibiotics to kill dormant organisms. The present study describes a system using Mycobacterium smegmatis, an avirulent mycobacterium, to deliver the lytic phage TM4 where both M. avium and M. tuberculosis reside within macrophages. These results showed that treatment of M. avium-infected, as well as M. tuberculosis-infected, RAW 264.7 macrophages, with M. smegmatis transiently infected with TM4, resulted in a significant time- and titer-dependent reduction in the number of viable intracellular bacilli. In addition, the M. smegmatis vacuole harboring TM4 fuses with the M. avium vacuole in macrophages. These results suggest a potentially novel concept to kill intracellular pathogenic bacteria and warrant future development.

NAD(H)-dependent l -alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination of l -alanine and the reductive amination of pyruvate. To assess the physiological role of Ald in Mycobacterium smegmatis , we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence of l - or d -alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with l -alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.

Mycobacterium paratuberculosis (MPT) is the etiologic agent of paratuberculosis. The disease is prevalent in cattle worldwide, and exacts a heavy financial toll. Effective control requires the development of acellular vaccines offering a better protection than the current available vaccines without side effects and allowing the discrimination between infected and vaccinated animals. We studied the immune response of mice to the MPT superoxide dismutase (SOD) alone or adjuvanted by Ribi. We cloned, overexpressed and purified this antigen in Escherichia coli. Spleen cells from immunized mice, after exposure to recombinant MPT SOD (MPT rSOD), produced significant levels of IFNgamma, TNFalpha and IL-6. IFNgamma and TNFalpha production was increased by the addition of Ribi. In contrast, low levels of NO, IL-4 and IL-10 were secreted by spleen cells culture from immunized mice. The immunoglobulin isotype distribution analysis showed that Ribi adjuvant clearly induced a significantly higher anti-MPT rSOD antibody production of all classes tested and decreased the IgG1/IgG2a ratio thus improving the Th1 response. Delayed-type hypersensitivity responses in mice footpads were observed only in mice immunized with MPT rSOD emulsified in Ribi. Vaccination of MPT rSOD emulsified with Ribi induced both a Th2 and Th1 type of immune response with the later slightly more pronounced. The results presented here on the immunogenicity of MPT SOD suggest that this antigen should be further tested as a candidate antigen for a future acellular vaccine against paratuberculosis.

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log 10 CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log 10 CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log 10 CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log 10 CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas , Clavibacter , Curtobacterium , and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum . These microorganisms may be useful for biocontrol and other applications.

Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the d -alanine racemase gene ( alrA ), which is involved in the synthesis of d -alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM d -alanine and 20 μg of kanamycin per ml. These mutants were also able to grow in standard and minimal media without d -alanine, giving rise to colonies with a drier appearance and more-raised borders than the wild-type strain. The viability of the mutants and independence of d -alanine for growth indicate that inactivation of alrA does not impose an auxotrophic requirement for d -alanine, suggesting the existence of a new pathway of d -alanine biosynthesis in M. smegmatis . Biochemical analysis demonstrated the absence of any detectable d -alanine racemase activity in the mutant strains. In addition, the alrA mutants displayed hypersusceptibility to the antimycobacterial agent d -cycloserine. The MIC of d -cycloserine for the mutant strain was 2.56 μg/ml, 30-fold less than that for the wild-type strain. Furthermore, this hypersusceptibility was confirmed by the bactericidal action of d -cycloserine on broth cultures. The kinetic of killing for the mutant strain followed the same pattern as that for the wild-type strain, but at a 30-fold-lower drug concentration. This effect does not involve a change in the permeability of the cell wall by this drug and is consistent with the identification of d -alanine racemase as a target of d -cycloserine. This outcome is of importance for the design of novel antituberculosis drugs targeting peptidoglycan biosynthesis in mycobacteria.

Since the early 1980s, several investigations have focused on developing a vaccine against Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease in cattle and sheep. These studies used whole-cell inactivated vaccines that have proven useful in limiting disease progression, but have not prevented infection. In contrast, modified live vaccines that invoke a Th1 type immune response, may improve protection against infection. Spurred by recent advances in the ability to create defined knockouts in MAP, several independent laboratories have developed modified live vaccine candidates by transpositional mutation of virulence and metabolic genes in MAP. In order to accelerate the process of identification and comparative evaluation of the most promising modified live MAP vaccine candidates, members of a multi-institutional USDA-funded research consortium, the Johne’s disease integrated program (JDIP), met to establish a standardized testing platform using agreed upon protocols. A total of 22 candidates vaccine strains developed in five independent laboratories in the United States and New Zealand voluntarily entered into a double blind stage gated trial pipeline. In Phase I, the survival characteristics of each candidate were determined in bovine macrophages. Attenuated strains moved to Phase II, where tissue colonization of C57/BL6 mice were evaluated in a challenge model. In Phase III, five promising candidates from Phase I and II were evaluated for their ability to reduce fecal shedding, tissue colonization and pathology in a baby goat challenge model. Formation of a multi-institutional consortium for vaccine strain evaluation has revealed insights for the implementation of vaccine trials for Johne’s disease and other animal pathogens. We conclude by suggesting the best way forward based on this 3-phase trial experience and challenge the rationale for use of a macrophage-to-mouse-to native host pipeline for MAP vaccine development.

Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which results in serious economic losses worldwide in farmed livestock such as cattle, sheep and goats. To control this disease, an effective vaccine with minimal adverse effects is needed. In order to identify a live vaccine for Johne’s disease, we evaluated eight attenuated mutant strains of MAP using a C57BL/6 mouse model. The persistence of the vaccine candidates was measured at 6, 12, and 18 weeks post vaccination. Only strains 320, 321 and 329 colonized both the liver and spleens up until the 12-week time point. The remaining five mutants showed no survival in those tissues, indicating their complete attenuation in the mouse model. The candidate vaccine strains demonstrated different levels of protection based on colonization of the challenge strain in liver and spleen tissues at 12 and 18 weeks post vaccination. Based on total MAP burden in both tissues at both time points, strain 315 (MAP1566::Tn5370) was the most protective whereas strain 318 (intergenic Tn5367 insertion between MAP0282c and MAP0283c) had the most colonization. Mice vaccinated with an undiluted commercial vaccine preparation displayed the highest bacterial burden as well as enlarged spleens indicative of a strong infection. Selected vaccine strains that showed promise in the mouse model were moved forward into a goat challenge model. The results suggest that the mouse trial, as conducted, may have a relatively poor predictive value for protection in a ruminant host such as goats.

Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a major threat to the dairy industry and possibly some cases of Crohn's disease in humans. A MAP vaccine that reduced of clinical disease and/or reduced fecal shedding would aid in the control of JD. The objectives of this study were (1) to evaluate the efficacy of 5 attenuated strains of MAP as vaccine candidates compared to a commercial control vaccine using the protocol proposed by the Johne's Disease Integrated Program (JDIP) Animal Model Standardization Committee (AMSC), and (2) to validate the AMSC Johne's disease goat challenge model. Eighty goat kids were vaccinated orally twice at 8 and 10 weeks of age with an experimental vaccine or once subcutaneously at 8 weeks with Silirum® (Zoetis), or a sham control oral vaccine at 8 and 10 weeks. Kids were challenged orally with a total of approximately 1.44 × 10(9) CFU divided in two consecutive daily doses using MAP ATCC-700535 (K10-like bovine isolate). All kids were necropsied at 13 months post-challenge. Results indicated that the AMSC goat challenge model is a highly efficient and valid model for JD challenge studies. None of the experimental or control vaccines evaluated prevented MAP infection or eliminated fecal shedding, although the 329 vaccine lowered the incidence of infection, fecal shedding, tissue colonization and reduced lesion scores, but less than the control vaccine. Based on our results the relative performance ranking of the experimental live-attenuated vaccines evaluated, the 329 vaccine was the best performer, followed by the 318 vaccine, then 316 vaccine, 315 vaccine and finally the 319 vaccine was the worst performer. The subcutaneously injected control vaccine outperformed the orally-delivered mutant vaccine candidates. Two vaccines (329 and 318) do reduce presence of JD gross and microscopic lesions, slow progression of disease, and one vaccine (329) reduced fecal shedding and tissue colonization.

Using a high throughput genetic strategy, designated Random Inducible Controlled Expression (RICE), we identified the six gene mel2 locus in Mtb and M. marinum. Interestingly, three of the genes present in mel2 have similarities to bioluminescence genes. Similar to other bacterial bioluminescence systems, mel2 facilitates detoxification of reactive oxygen species (ROS). Through the use of thin layer chromatography (TLC) we demonstrate enhanced production of the cell wall virulence lipid, pthiocerol dimycoserosate (PDIM), in a Mtb mel2 mutant relative to the wild type strain in the presence of both H2O2 and diamide oxidative stresses. Furthermore, propionate toxicity assays revealed increased accumulation of triacylglycerol (TAG) in the mel2 mutant relative to wild type. These observations provide the first evidence that mel2 plays a critical role in Mtb lipid biosynthesis.

Escherichia coli K-12 carries the cryptic hemolysin gene sheA which is under the control of positive and negative transcriptional regulators. The objectives of the present study were to further analyze the regulation of the sheA gene in E. coli, to compare the sheA genes from E. coli K-12 and a pathogenic E. coli strain, and to characterize the SheA hemolytic activity. Northern blot analysis demonstrated that the transcriptional regulator SlyA activates the E. coli K-12 sheA gene. The main transcriptional start site of the sheA gene was 56 nucleotides upstream from the start codon as determined by primer extension analysis. The sheA genes from E. coli K-12 and a pathogenic E. coli strain were identical. SheA hemolytic activity was cell associated and Ca2+ independent.

Pigs infected with hemolytic F4 + strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4 + enterotoxigenic E. coli to cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene ( hlyA ) did not decrease the incidence of septicemia in the gnotobiotic piglet model.

Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry.

Over the past decade, metabolomics has emerged as an important technique for systems biology. Measuring all the metabolites in a biological system provides an invaluable source of information to explore various cellular processes, and to investigate the impact of environmental factors and genetic modifications. Nuclear magnetic resonance (NMR) spectroscopy is an important method routinely employed in metabolomics. NMR provides comprehensive structural and quantitative information useful for metabolomics fingerprinting, chemometric analysis, metabolite identification and metabolic pathway construction. A successful metabolomics study relies on proper experimental protocols for the collection, handling, processing and analysis of metabolomics data. Critically, these protocols should eliminate or avoid biologically-irrelevant changes to the metabolome. We provide a comprehensive description of our NMR-based metabolomics procedures optimized for the analysis of bacterial metabolomes. The technical details described within this manuscript should provide a useful guide to reliably apply our NMR-based metabolomics methodology to systems biology studies.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment, 1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 °C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1 H NMR and two-dimensional (2D) 1 H- 13 C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

The traditional diagnostic tests for tuberculosis consist of an acid fast stain and a culture test from a sputum sample. With the emergence of drug resistant strains of tuberculosis, nucleic acid amplification has become the diagnostic test of choice. The nucleic acid amplification test consists of four steps: sputum sample collection, lysis of bacilli to release DNA, DNA amplification by PCR and detection of PCR products. The DNA extraction step has been largely overlooked and this study describes a systematic approach to measure the kinetics of cell lysis in a Tris-EDTA buffer. Mycobacterium smegmatis is a saphorytic, fast-growing mycobacterium that is often used as a surrogate of Mycobacterium tuberculosis in laboratory studies. M. smegmatis cells have been transformed with green fluorescent protein (GFP) genes. Transformed cells are lysed in a temperature-controlled cuvette that is equipped with optical input/output. The fluorescence signal increases when the GFP is released from lysed cells, and the extent of lysis of the loaded cells can be followed in real time. The experimental results are complemented by two theoretical models. The first model is based on a Monte Carlo simulation of the lysis process and the accompanying probability density function as described by the Fokker-Planck equation. The second model follows a chemical reaction engineering approach: the cell wall is modeled as layers, where each layer is made up of ‘blocks’. Blocks can only be removed if they are exposed to the lysis solution and the model describes the rate of block exposure and removal. Both models are consistent with the experimental results. The main findings are: (1) the activation energy for M. smegmatis lysis by Tris-EDTA buffer is 22.1kcal/mole, (2) cells lyse on the average after 14–17% loss in cell wall thickness locally, (3) with the help of the models, the initial distribution in cell wall thickness of the population can be resolved, (4) near complete lysis of the cells is accomplished in 200 seconds at 80°C (90 seconds at 90°C). The results can be used to design an optimal lysis protocol that compromises between shorter processing times at higher temperature and reduced thermal damage to DNA at lower temperature.

Effective chemotherapy of tuberculosis requires rapid assessment of drug sensitivity because of the emergence of multidrug-resistant Mycobacterium tuberculosis. Drug susceptibility was assessed by a simple method based on the efficient production of photons by viable mycobacteria infected with specific reporter phages expressing the firefly luciferase gene. Light production was dependent on phage infection, expression of the luciferase gene, and the level of cellular adenosine triphosphate. Signals could be detected within minutes after infection of virulent M. tuberculosis with reporter phages. Culture of conventional strains with antituberculosis drugs, including isoniazid or rifampicin, resulted in extinction of light production. In contrast, light signals after luciferase reporter phage infection of drug-resistant strains continued to be produced. Luciferase reporter phages may help to reduce the time required for establishing antibiotic sensitivity of M. tuberculosis strains from weeks to days and to accelerate screening for new antituberculosis drugs.

Mycobacterium aviumsubsp.paratuberculosis, the agent of Johne's disease, infects ruminant hosts by translocation through the intestinal mucosa. A number of studies have suggested thatM. aviumsubsp.paratuberculosisinteracts with M cells in the Peyer's patches of the small intestine. The invasion of the intestinal mucosa byM. aviumsubsp.paratuberculosisandMycobacterium aviumsubsp.hominissuis, a pathogen known to interact with intestinal cells, was compared.M. aviumsubsp.paratuberculosiswas capable of invading the mucosa, but it was significantly less efficient at dissemination thanM. aviumsubsp.hominissuis. B-cell knockout (KO) mice, which lack Peyer's patches, were used to demonstrate thatM. aviumsubsp.paratuberculosisenters the intestinal mucosa through enterocytes in the absence of M cells. In addition, the results indicated thatM. aviumsubsp.paratuberculosishad equal abilities to cross the mucosa in both Peyer's patch and non-Peyer's patch segments of normal mice.M. aviumsubsp.paratuberculosiswas also shown to interact with epithelial cells by an α5β1integrin-independent pathway. Upon translocation, dendritic cells ingestM. aviumsubsp.paratuberculosis, but this process does not lead to efficient dissemination of the infection. In summary,M. aviumsubsp.paratuberculosisinteracts with the intestinal mucosa by crossing both Peyer's patches and non-Peyer's patch areas but does not translocate or disseminate efficiently.