The crystal structure of the mycobacterial trehalose monomycolate transport factor A, TtfA, reveals an atypical fold.

Trehalose monomycolate (TMM) represents an essential element of the mycobacterial envelope. While synthesized in the cytoplasm, TMM is transported across the inner membrane by MmpL3 but, little is known regarding the MmpL3 partners involved in this process. Recently, the TMM transport factor A (TtfA) was found to form a complex with MmpL3 and to participate in TMM transport, although its biological role remains to be established. Herein, we report the crystal structure of the Mycobacterium smegmatis TtfA core domain. The phylogenetic distribution of TtfA homologues in non-mycolate containing bacteria suggests that TtfA may exert additional functions.

INNO-LiPA DNA line probe assay misidentification of M. smegmatis as Mycobacterium fortuitum complex.

Seven weeks after being kicked in the face by a cow, a 34-year-old male patient developed a posttraumatic mycobacterial lymphadenitis. A rapidly growing mycobacterial isolate cultured from a surgically drained lymphadenitis pus specimen was identified as Mycobacterium smegmatis by matrix-assisted laser desorption/ionization mass spectrometry and a combination of ITS-, hsp65-, and 16S rRNA-DNA sequence analysis, but as Mycobacterium fortuitum complex using the commercial INNO-LiPA Mycobacteria v2 line probe assay. As it is unclear if the misidentification of this strain is an exception, more research is required.

Identifying Dormant Growth State of Mycobacteria by Orthogonal Analytical Approaches on a Single Cell and Ensemble Basis.

Tuberculosis is currently the single most deadly infectious disease in the world and a public health priority as defined by WHO. Although the disease is in general curable, treatment success is hampered by the necessity of a long and side effect prone treatment. Low treatment efficiency may be partly due to the special growth states that mycobacteria enter to avoid being killed by antibiotics and to persist longer within the host. Such growth states have been recently defined as dormant or persistent. We produced dormant model-organism cultures using an acidification model and characterized those by a multilayered approach using mass spectrometry (MALDI-TOF), microscopy (SEM, Raman), and microbiological techniques (CFU, OD600, ATP-levels). With a fast and 96-well-adapted extraction protocol, mycobacteria could be inactivated and extracted for MALDI-TOF analysis. For the first time, we demonstrate growth-state-dependent changes in the mass signatures of the culture, allowing for a reliable differentiation of dormant state and exponential growth. We also demonstrate resuscitation from dormant state back to exponential growth. Viable mycobacteria were immobilized, and single organisms were analyzed individually by Raman microscopy. For single-cell Raman microscopy, Mycobacterium smegmatis cultures were fixed using a new fast and gentle single-step immobilization technique on a hydrophobic glass slide. We were able to distinguish single viable bacteria in the dormant state from their rapidly growing, genetically identical counterparts, identifying the growth state of the culture based on single-organism spectra. This allows for the separation of heterogeneous cultures depending on their growth state using the destruction-free optical method of Raman microscopy.

A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides.

BACKGROUND: The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target. RESULTS: Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM. CONCLUSIONS: This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system.

Eis, a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87).

Enhanced intracellular survival (Eis) proteins were found to enhance the intracellular survival of mycobacteria in macrophages by acetylating aminoglycoside antibiotics to confer resistance to these antibiotics and by acetylating DUSP16/MPK-7 to suppress host innate immune defenses. Eis homologs composing of two GCN5 N-acetyltransferase regions and a sterol carrier protein fold are found widely in gram-positive bacteria. In this study, we found that Eis proteins have an unprecedented ability to acetylate many arylalkylamines, are a novel type of arylalkylamine N-acetyltransferase AANAT (EC 2.3.1.87). Sequence alignment and phyletic distribution analysis confirmed Eis belongs to a new aaNAT-like cluster. Among the cluster, we studied three typical Eis proteins: Eis_Mtb from Mycobacterium tuberculosis, Eis_Msm from Mycobacterium smegmatis, and Eis_Sen from Saccharopolyspora erythraea. Eis_Mtb prefers to acetylate histamine and octopamine, while Eis_Msm uses tyramine and octopamine as substrates. Unlike them, Eis_Sen exihibits good catalytic efficiencies for most tested arylalkylamines. Considering arylalkylamines such as histamine plays a fundamental role in immune reactions, future work linking of AANAT activity of Eis proteins to their physiological function will broaden our understanding of gram-positive pathogen-host interactions. These findings shed insights into the molecular mechanism of Eis, and reveal potential clinical implications for many gram-positive pathogens.

Characterization of mycobacteria and mycobacteriophages isolated from compost at the São Paulo Zoo Park Foundation in Brazil and creation of the new mycobacteriophage Cluster U.

BACKGROUND: A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo. RESULTS: We isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc(2)155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system. CONCLUSIONS: We predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution.

Magnetic Multivalent Trehalose Glycopolymer Nanoparticles for the Detection of Mycobacteria.

A multivalent trehalose-grafted poly(lactic acid) is synthesized and encapsulated with iron oxide magnetic nanoparticles. The magnetic micelles interact with Mycobacterium smegmatis to form orange clusters. Very little particle interaction is found on Staphylococcus epidermidis 35984 or Escherichia coli ORN 208. The presented new approach to the detection of mycobacteria does not require molecular biology reagents or sophisticated instruments.

Bacterial Suspensions Deposited on Microbiological Filter Material for Rapid Laser-Induced Breakdown Spectroscopy Identification.

Four species of bacteria, E. coli, S. epidermidis, M. smegmatis, and P. aeruginosa, were harvested from agar nutrient medium growth plates and suspended in water to create liquid specimens for the testing of a new mounting protocol. Aliquots of 30 µL were deposited on standard nitrocellulose filter paper with a mean 0.45 µm pore size to create highly flat and uniform bacterial pads. The introduction of a laser-based lens-to-sample distance measuring device and a pair of matched off-axis parabolic reflectors for light collection improved both spectral reproducibility and the signal-to-noise ratio of optical emission spectra acquired from the bacterial pads by laser-induced breakdown spectroscopy. A discriminant function analysis and a partial least squares-discriminant analysis both showed improved sensitivity and specificity compared to previous mounting techniques. The behavior of the spectra as a function of suspension concentration and filter coverage was investigated, as was the effect on chemometric cell classification of sterilization via autoclaving.

Structural characterization of a mitochondrial 3-ketoacyl-CoA (T1)-like thiolase from Mycobacterium smegmatis.

Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) from Mycobacterium smegmatis in apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domain via flexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The Lα1-covering loop-Lα2 region, together with the Nß2-Nα2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains.

Characteristics of Mycobacterium smegmatis J15cs strain lipids.

Mycobacterium smegmatis is a rapidly growing, non-pathogenic mycobacterium, and M. smegmatis strain mc(2)155 in particular has been used as a tool for molecular analysis of mycobacteria because of its high rate of transformation. We examined another strain, M. smegmatis J15cs, which has the advantage of surviving for six days in murine macrophages. The J15cs strain produces a rough dry colony, and we hypothesized that the long survival of the J15cs strain was correlated with its cell wall components. Therefore, the lipid compositions of these two strains were compared. The subclasses and carbon species of the mycolic acids were very similar, and the major glycolipids and phospholipids were expressed in both strains. However, apolar glycopeptidolipids were deleted only in the J15cs strain. The presence of apolar glycopeptidolipids gives the cell wall a different structure. Moreover, the apolar glycopeptidolipids were recognized by macrophages via toll-like receptor 2, but not 4. We concluded that the absence of apolar glycopeptidolipids is a definitive feature of the J15cs strain, and affects its morphology and survival in host cells.

Ability of Cricetomys rats to detect Mycobacterium tuberculosis and discriminate it from other microorganisms.

Trained African giant pouched rats (Cricetomys gambianus) have potential for diagnosis of tuberculosis (TB). These rats target volatile compounds of Mycobacterium tuberculosis (Mtb) that cause TB. Mtb and nontuberculous mycobacteria (NTM) species are related to Nocardia and Rhodococcus spp., which are also acid-fast bacilli and can be misdiagnosed as Mtb in smear microscopy. Diagnostic performance of C. gambianus on in vitro-cultured mycobacterial and related pulmonary microbes is unknown. This study reports on the response of TB detection rats to cultures of reference Mtb, clinical Mtb, NTM, Nocardia; Rhodococcus; Streptomyces; Bacillus; and yeasts. Trained rats significantly discriminated Mtb from other microbes (p < 0.008, Fisher's exact test). Detection of Mtb cultures was age-related, with exponential and early stationary phase detected more frequently than early log phase and late stationary phase (p < 0.001, Fisher's test) (sensitivity = 83.33%, specificity = 94.4%, accuracy = 94%). The detection of naturally TB-infected sputum exceeded that of negative sputum mixed with Mtb, indicating that C. gambianus are conditioned to detect odours of TB-positive sputum better than spiked sputum. Although further studies on volatiles from detectable growth phases of Mtb are vital for identification of Mtb-specific volatiles detected by rats, our study underline the potential of C. gambianus for TB diagnosis.

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria.

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+ fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.

Antigen 84, an effector of pleiomorphism in Mycobacterium smegmatis.

While in most rod-shaped bacteria, morphology is based on MreB-like proteins that form an actin-like cytoskeletal scaffold for cell wall biosynthesis, the factors that determine the more flexible rod-like shape in actinobacteria such as Mycobacterium species are unknown. Here we show that a Mycobacterium smegmatis protein homologous to eubacterial DivIVA-like proteins, including M. tuberculosis antigen 84 (Ag84), localized symmetrically to centers of peptidoglycan biosynthesis at the poles and septa. Controlled gene disruption experiments indicated that the gene encoding Ag84, wag31, was essential; when overexpressed, cells became longer and wider, with Ag84 asymmetrically distributed at one pole. Many became grossly enlarged, bowling-pin-shaped cells having up to 80-fold-increased volume. In these cells, Ag84 accumulated predominantly at a bulbous pole that was apparently generated by uncontrolled cell wall expansion. In some cells, Ag84 was associated with exceptional sites of cell wall expansion (buds) that evolved into branches. M. bovis BCG Ag84 was able to form oligomers in vitro, perhaps reflecting its superstructure in vivo. These data suggested a role for Ag84 in cell division and modulating cell shape in pleiomorphic actinobacteria.

Analysis of mycobacterial strains by two-dimensional gel electrophoresis.

This study pertains to analysis of the protein profile of different mycobacterial strains by two-dimensional gel electrophoresis (2DE). The strains were selected as they exhibit different phenotypic behaviour. TCA-acetone precipitated proteins were resolved by 2DE using immobilized pH gradient (IPG) strips. This study demonstrates that 2DE may be used as a tool for characterization of mycobacterial strains. Visual examination of the electrophoretograms was sufficient for characterization. Detailed characterization of specific proteins might lead to development of novel targets, diagnostic probes or sub-unit vaccine(s) against tuberculosis.

Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing.

The current classification of non-pigmented and late-pigmenting rapidly growing mycobacteria (RGM) capable of producing disease in humans and animals consists primarily of three groups, the Mycobacterium fortuitum group, the Mycobacterium chelonae-abscessus group and the Mycobacterium smegmatis group. Since 1995, eight emerging species have been tentatively assigned to these groups on the basis of their phenotypic characters and 16S rRNA gene sequence, resulting in confusing taxonomy. In order to assess further taxonomic relationships among RGM, complete sequences of the 16S rRNA gene (1483-1489 bp), rpoB (3486-3495 bp) and recA (1041-1056 bp) and partial sequences of hsp65 (420 bp) and sodA (441 bp) were determined in 19 species of RGM. Phylogenetic trees based upon each gene sequence, those based on the combined dataset of the five gene sequences and one based on the combined dataset of the rpoB and recA gene sequences were then compared using the neighbour-joining, maximum-parsimony and maximum-likelihood methods after using the incongruence length difference test. Combined datasets of the five gene sequences comprising nearly 7000 bp and of the rpoB+recA gene sequences comprising nearly 4600 bp distinguished six phylogenetic groups, the M. chelonae-abscessus group, the Mycobacterium mucogenicum group, the M. fortuitum group, the Mycobacterium mageritense group, the Mycobacterium wolinskyi group and the M. smegmatis group, respectively comprising four, three, eight, one, one and two species. The two protein-encoding genes rpoB and recA improved meaningfully the bootstrap values at the nodes of the different groups. The species M. mucogenicum, M. mageritense and M. wolinskyi formed new groups separated from the M. chelonae-abscessus, M. fortuitum and M. smegmatis groups, respectively. The M. mucogenicum group was well delineated, in contrast to the M. mageritense and M. wolinskyi groups. For phylogenetic organizations derived from the hsp65 and sodA gene sequences, the bootstrap values at the nodes of a few clusters were <70 %. In contrast, phylogenetic organizations obtained from the 16S rRNA, rpoB and recA genes were globally similar to that inferred from combined datasets, indicating that the rpoB and recA genes appeared to be useful tools in addition to the 16S rRNA gene for the investigation of evolutionary relationships among RGM species. Moreover, rpoB gene sequence analysis yielded bootstrap values higher than those observed with recA and 16S rRNA genes. Also, molecular signatures in the rpoB and 16S rRNA genes of the M. mucogenicum group showed that it was a sister group of the M. chelonae-abscessus group. In this group, M. mucogenicum ATCC 49650(T) was clearly distinguished from M. mucogenicum ATCC 49649 with regard to analysis of the five gene sequences. This was in agreement with phenotypic and biochemical characteristics and suggested that these strains are representatives of two closely related, albeit distinct species.

Characterization of a novel rapidly growing Mycobacterium species associated with sepsis.

A rapidly growing mycobacterium was isolated five times from blood cultures from a 6-year-old female patient with relapsed pre-B-cell acute lymphocytic leukemia. All five isolates had identical nucleotide sequences for the first 500 bp of the 16S rRNA gene, indicative of a single species. High-performance liquid chromatography analysis of mycolic acids indicated that the species was similar to Mycobacterium smegmatis. Sequence analysis of the 16S rRNA gene (1,455 bp) for one isolate demonstrated that the species was closely related to Mycobacterium diernhoferi. Based on the phenotypic features and phylogenetic analysis, it was concluded that the isolates represented a novel rapidly growing Mycobacterium species. The name "Mycobacterium hackensackense" is proposed for this unique strain, 147-0552(T), which was deposited in the American Type Culture Collection as ATCC BAA-823(T).

Molecular and culture-based analyses of aerobic carbon monoxide oxidizer diversity.

Isolates belonging to six genera not previously known to oxidize CO were obtained from enrichments with aquatic and terrestrial plants. DNA from these and other isolates was used in PCR assays of the gene for the large subunit of carbon monoxide dehydrogenase (coxL). CoxL and putative coxL fragments were amplified from known CO oxidizers (e.g., Oligotropha carboxidovorans and Bradyrhizobium japonicum), from novel CO-oxidizing isolates (e.g., Aminobacter sp. strain COX, Burkholderia sp. strain LUP, Mesorhizobium sp. strain NMB1, Stappia strains M4 and M8, Stenotrophomonas sp. strain LUP, and Xanthobacter sp. strain COX), and from several well-known isolates for which the capacity to oxidize CO is reported here for the first time (e.g., Burkholderia fungorum LB400, Mesorhizobium loti, Stappia stellulata, and Stappia aggregata). PCR products from several taxa, e.g., O. carboxidovorans, B. japonicum, and B. fungorum, yielded sequences with a high degree (>99.6%) of identity to those in GenBank or genome databases. Aligned sequences formed two phylogenetically distinct groups. Group OMP contained sequences from previously known CO oxidizers, including O. carboxidovorans and Pseudomonas thermocarboxydovorans, plus a number of closely related sequences. Group BMS was dominated by putative coxL sequences from genera in the Rhizobiaceae and other alpha-PROTEOBACTERIA: PCR analyses revealed that many CO oxidizers contained two coxL sequences, one from each group. CO oxidation by M. loti, for which whole-genome sequencing has revealed a single BMS-group putative coxL gene, strongly supports the notion that BMS sequences represent functional CO dehydrogenase proteins that are related to but distinct from previously characterized aerobic CO dehydrogenases.

Clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria.

The history, taxonomy, geographic distribution, clinical disease, and therapy of the pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria (RGM) are reviewed. Community-acquired disease and health care-associated disease are highlighted for each species. The latter grouping includes health care-associated outbreaks and pseudo-outbreaks as well as sporadic disease cases. Treatment recommendations for each species and type of disease are also described. Special emphasis is on the Mycobacterium fortuitum group, including M. fortuitum, M. peregrinum, and the unnamed third biovariant complex with its recent taxonomic changes and newly recognized species (including M. septicum, M. mageritense, and proposed species M. houstonense and M. bonickei). The clinical and taxonomic status of M. chelonae, M. abscessus, and M. mucogenicum is also detailed, along with that of the closely related new species, M. immunogenum. Additionally, newly recognized species, M. wolinskyi and M. goodii, as well as M. smegmatis sensu stricto, are included in a discussion of the M. smegmatis group. Laboratory diagnosis of RGM using phenotypic methods such as biochemical testing and high-performance liquid chromatography and molecular methods of diagnosis are also discussed. The latter includes PCR-restriction fragment length polymorphism analysis, hybridization, ribotyping, and sequence analysis. Susceptibility testing and antibiotic susceptibility patterns of the RGM are also annotated, along with the current recommendations from the National Committee for Clinical Laboratory Standards (NCCLS) for mycobacterial susceptibility testing.

Mycobacterium wolinskyi sp. nov. and Mycobacterium goodii sp. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections: a cooperative study from the International Working Group on Mycobacterial Taxonomy.

Previous investigations demonstrated three taxonomic groups among 22 clinical isolates of Mycobacterium smegmatis. These studies were expanded to 71 clinical isolates, of which 35 (49%) (group 1) were identical to five ATCC reference strains including the type strain ATCC 19420T. Twenty-eight isolates (39%) were group 2, and eight isolates (11%) were group 3. Isolates of groups 2 and 3 were most often associated with post-traumatic or post-surgical wound infections including osteomyelitis, were susceptible to sulfamethoxazole, amikacin, imipenem and the tetracyclines, variably resistant to clarithromycin, and susceptible (group 1), intermediately resistant (group 2) or resistant (group 3) to tobramycin. The three groups were similar by routine biochemical and growth characteristics, but had different mycolic acid dimethoxy-4-coumarinylmethyl ester elution patterns by HPLC and different PCR-restriction enzyme patterns of a 439 bp fragment of the hsp-65 gene. Group 3 isolates differed from group 1 by 18 bp by 16S rRNA sequencing and exhibited < 25% homology by DNA-DNA hybridization, being most closely related to Mycobacterium mageritense. The 16S rRNA of group 1 and group 2 isolates differed by only 3 bp, but by DNA-DNA hybridization they exhibited only 40% homology. The following names are proposed: Mycobacterium goodii sp. nov. for group 2 isolates (type strain ATCC 700504T = MO69T), Mycobacterium wolinskyi sp. nov. for group 3 isolates (type strain ATCC 700010T = MO739T) and Mycobacterium smegmatis sensu stricto for group 1 isolates.