Severity of Bovine Tuberculosis Is Associated with Co-Infection with Common Pathogens in Wild Boar

Co-infections with parasites or viruses drive tuberculosis dynamics in humans, but little is known about their effects in other non-human hosts. This work aims to investigate the relationship between Mycobacterium bovis infection and other pathogens in wild boar (Sus scrofa), a recognized reservoir of bovine tuberculosis (bTB) in Mediterranean ecosystems. For this purpose, it has been assessed whether contacts with common concomitant pathogens are associated with the development of severe bTB lesions in 165 wild boar from mid-western Spain. The presence of bTB lesions affecting only one anatomic location (cervical lymph nodes), or more severe patterns affecting more than one location (mainly cervical lymph nodes and lungs), was assessed in infected animals. In addition, the existence of contacts with other pathogens such as porcine circovirus type 2 (PCV2), Aujeszky''s disease virus (ADV), swine influenza virus, porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and Metastrongylus spp, was evaluated by means of serological, microbiological and parasitological techniques. The existence of contacts with a structured community of pathogens in wild boar infected by M. bovis was statistically investigated by null models. Association between this community of pathogens and bTB severity was examined using a Partial Least Squares regression approach. Results showed that adult wild boar infected by M. bovis had contacted with some specific, non-random pathogen combinations. Contact with PCV2, ADV and infection by Metastrongylus spp, was positively correlated to tuberculosis severity. Therefore, measures against these concomitant pathogens such as vaccination or deworming, might be useful in tuberculosis control programmes in the wild boar. However, given the unexpected consequences of altering any community of organisms, further research should evaluate the impact of such measures under controlled conditions. Furthermore, more research including other important pathogens, such as gastro-intestinal nematodes, will be necessary to complete this picture.     

Spatio-Temporal Trends of Iberian Wild Boar Contact with Mycobacterium tuberculosis Complex Detected by ELISA

The continuing expansion of Eurasian wild boar (Sus scrofa) populations raises concerns regarding disease transmission. In south-central Spain, overabundant wild boar are reservoirs of Mycobacterium bovis, and related members of the Mycobacterium tuberculosis complex (MTBC), the causative agents of bovine tuberculosis (bTB). An indirect enzyme-linked immunosorbent assay using bovine-purified protein derivative was applied to determine the spatial and temporal distribution of wild boar contact with MTBC in the Iberian Peninsula and to model and identify the associated risk factors. Wild boar apparent seroprevalence was 22%. Seropositives were detected in 71% of 81 sites, including 23 sites where wildlife was thought to be bTB free. The results described a new geographic range of wild boar contact with MTBC and a stable prevalence in this wildlife reservoir that contrasts with the success of bTB control in cattle. Inference of which host (wild boar or cattle) is driving bTB maintenance was not possible with our correlational results. The possibility of a wild boar bTB emergence in non-endemic regions should urgently be taken into account to avoid a future scenario resembling the current situation in south-central Spain.     

Role of Cattle Movements in Bovine Tuberculosis Spread in France between 2005 and 2014

Live animal movements are a major transmission route for the spread of infectious agents such as Mycobacterium bovis, the main agent of bovine Tuberculosis (bTB). France became officially bTB-free in 2001, but M. bovis is still circulating in the cattle population, with about a hundred of outbreaks per year, most located in a few geographic areas. The aim of this study was to analyse the role of cattle movements in bTB spread in France between 2005 and 2014, using social network analysis and logistic regression models. At a global scale, the trade network was studied to assess the association between several centrality measures and bTB infection though a case-control analysis. The bTB infection status was associated with a higher in-degree (odds-ratio [OR] = 2.4 [1.1–5.4]) and with a higher ingoing contact chain (OR = 2.2 [1.0–4.7]). At a more local scale, a second case-control analysis was conducted to estimate the relative importance of cattle movements and spatial neighbourhood. Only direct purchase from infected herds was shown to be associated with bTB infection (OR = 2.9 [1.7–5.2]), spatial proximity to infected herds being the predominant risk factor, with decreasing ORs when distance increases. Indeed, the population attributable fraction was 12% [5%–18%] for cattle movements and 73% [68%–78%] for spatial neighbourhood. Based on these results, networks of potential effective contacts between herds were built and analysed for the three major spoligotypes reported in France. In these networks, the links representing cattle movements were associated with higher edge betweenness than those representing the spatial proximity between infected herds. They were often links connecting distinct communities and sometimes distinct geographical areas. Therefore, although their role was quantitatively lower than the one of spatial neighbourhood, cattle movements appear to have been essential in the French bTB dynamics between 2005 and 2014.     

Genetic Evolution of Mycobacterium bovis Causing Tuberculosis in Livestock and Wildlife in France since 1978

To study the dynamics of bovine tuberculosis (bTB) in France, 4,654 M. bovis strains isolated mainly from livestock and wildlife since 1978 were characterized by spoligotyping and MLVA based on MIRU-VNTR. In our study spoligotyping allowed the discrimination of 176 types although 3 spoligotypes are predominant and account for more than half of the total strain population: SB0120 (26%), SB0134 (11%) and SB0121 (6%). In addition, 11% of the isolates, principally from Southern France, showing close spoligotypes and MIRU-VNTR types have been gathered in a family designated as the “F4-family”. MLVA typing allowed extensive discrimination, particularly for strains with predominant spoligotypes, with a total of 498 genotypes, several of which were highly regionalized. The similarity of the strains’ genetic relationships based on spoligotyping and MIRU-VNTR markers supports the co-existence of different clonal populations within the French M. bovis population. A genetic evolution of the strains was observed both geographically and in time. Indeed, as a result of the reduction of bTB due to the national control campaigns, a large reduction of the strains’ genetic variability took place in the last ten years. However, in the regions were bTB is highly prevalent at present, cases in both livestock and in wildlife are due to the spread of unique local genotype profiles. Our results show that the highly discriminating genotyping tools used in this study for molecular studies of bTB are useful for addressing pending questions, which would lead to a better insight into the epidemiology of the disease, and for finding proper solutions for its sustainable control in France.     

Spatial Ecology of Raccoons Related to Cattle and Bovine Tuberculosis in Northeastern Michigan

ABSTRACT In 1995, Mycobacterium bovis, the causative bacterium of bovine tuberculosis (bTB), was detected in 5 beef cattle operations in Alcona County, Michigan, USA. In accordance with Federal law, the operations were depopulated to prevent the spread of bTB. Subsequent wildlife surveillance programs identified high prevalence of M. bovis in mesocarnivores, including raccoons (Procyon lotor), which suggested that raccoons may be complicit in vectoring the pathogen among livestock operations. Our goal was to develop an empirical basis for generating hypotheses about the likelihood for raccoons to mediate the transmission of bTB to livestock. We found intersexual differences in scale-dependent resource selection and probability of spatial interaction that, under certain circumstances, may form the foundation for a sex-bias in disease transmission. Spatial dispersion of mixed-forest patches facilitated overlap of adjacent males, whereas female overlap zones included pastures. Within overlap zones, probabilities of interaction for male-male and male-female dyads were greater than for female-female dyads, although we documented an elevated likelihood of spatial interaction between raccoons and livestock around cattle-feeding troughs and water sources, regardless of sex. Partial regressions generated by linear models indicated that distance between nearest-neighbor mixed-forest patches explained most of this observed variation. These results supported our prediction that forest patches juxtaposed with anthropogenic features fostered social tolerance between males and, thus, facilitated spatial interaction and exploitation of anthropogenic features. In raccoons, sex and landscape composition influenced pathogen transmission potential. We suggest that livestock producers locate livestock feeding and watering features away from forest patches to mitigate future outbreaks of bTB in endemic areas.     

Revisiting Host Preference in the Mycobacterium tuberculosis Complex: Experimental Infection Shows M. tuberculosis H37Rv to Be Avirulent in Cattle

Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 10⁶ CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-γ and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97–infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis–infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis of host preference, and in particular the molecular basis of virulence in M. bovis.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

Characterization of Effector and Memory T Cell Subsets in the Immune Response to Bovine Tuberculosis in Cattle

Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7⁺, CD62L^hi, CD45RO⁺), T effector memory (Tem, defined as: CCR7^-, CD62L^low/int, CD45RO⁺), and T effector cells (CCR7^-, CD62L^-/low, CD45RO^-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4⁺ IFN-γ⁺ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.     

Diagnosis of Tuberculosis in the Wild Boar (Sus scrofa): A Comparison of Methods Applicable to Hunter-Harvested Animals

Background

To obtain robust epidemiological information regarding tuberculosis (TB) in wildlife species, appropriate diagnostic methods need to be used. Wild boar (Sus scrofa) recently emerged as a major maintenance host for TB in some European countries. Nevertheless, no data is available to evaluate TB post-mortem diagnostic methods in hunter-harvested wild boar.

Methodology/Principal Findings

Six different diagnostic methods for TB were evaluated in parallel in 167 hunter-harvested wild boar. Compared to bacteriological culture, estimates of sensitivity of histopathology was 77.8%, gross pathology 72.2%, PCR for the MPB70 gene 66.7%, detection of acid-fast bacilli (AFB) in tissue contact smears 55.6% and in histopathology slides 16.7% (estimated specificity was 96.7%, 100%, 100%, 94.4% and 100%, respectively). Combining gross pathology with stained smears in parallel increased estimated sensitivity to 94.4% (94.4% specificity). Four probable bacteriological culture false-negative animals were identified by Discriminant Function Analysis. Recalculating the parameters considering these animals as infected generated estimated values for sensitivity of bacteriology and histopathology of 81.8%, gross pathology 72.7%, PCR for the MPB70 gene 63.6%, detection of AFB in tissue contact smears 54.5% and in histopathology slides 13.6% (estimated specificity was 100% for gross pathology, PCR, bacteriology and detection of AFB in histopathology slides, 96.7% for histopathology and 94.4% for stained smears).

Conclusions/Significance

These results show that surveys for TB in wild boar based exclusively on gross pathology considerably underestimate prevalence, while combination of tests in parallel much improves sensitivity and negative predictive values. This finding should thus be considered when planning future surveys and game meat inspection schemes. Although bacteriological culture is the reference test for TB diagnosis, it can generate false-negative results and this should be considered when interpreting data.     

The Defect in Autophagy Induction by Clinical Isolates of Mycobacterium Tuberculosis Is Correlated with Poor Tuberculosis Outcomes

Background

Tuberculosis (TB) represents a major global health problem. The prognosis of clinically active tuberculosis depends on the complex interactions between Mycobacterium tuberculosis (Mtb) and its host. In recent years, autophagy receives particular attention for its role in host defense against intracellular pathogens, including Mtb. In present study, we aim to investigate the relationship of autophagy induction by clinical isolates of Mtb with the clinical outcomes in patients with TB.

Methodology/Principal Findings

We collected 185 clinical isolates of Mtb, and determined the effect of these Mtb isolates on autophagy induction in macrophages. It was found that most of clinical isolates of Mtb were able to induce autophagosome formation in macrophages, however, the autophagy-inducing ability varied significantly among different isolates. Of importance, our results revealed that patients infected by Mtb with poor autophagy-inducing ability displayed more severe radiographic extent of disease (p<0.001), and were more likely to have unfavorable treatment outcomes (p<0.001). No significant association was observed between the extent of Mtb-induced autophagy with some socio-demographic characteristics (such as gender, age and tobacco consumption), and some laboratory tests (such as hemoglobin, leukocyte count and erythrocyte sedimentation rate). Furthermore, results from logistic regression analysis demonstrated that the defect in autophagy induction by clinical isolates of Mtb was an independent risk factor for far-advanced radiographic disease (aOR 4.710 [1.93–11.50]) and unfavorable treatment outcomes (aOR 8.309 [2.22–28.97]) in TB.

Conclusion/Significance

These data indicated that the defect in autophagy induction by Mtb isolates increased the risk of poor clinical outcomes in TB patients, and detection of clinical isolates-induced autophagosome formation might help evaluate the TB outcomes.     

Bovine tuberculosis (Mycobacterium bovis) in British farmland wildlife: the importance to agriculture

Bovine tuberculosis (bTB) is an important disease of cattle and an emerging infectious disease of humans. Cow- and badger-based control strategies have failed to eradicate bTB from the British cattle herd, and the incidence is rising by about 18%per year. The annual cost to taxpayers in Britain is currently 74 million UK pounds. Research has focused on the badger as a potential bTB reservoir, with little attention being paid to other mammals common on farmland. We have conducted a systematic survey of wild mammals (n=4393 individuals) present on dairy farms to explore the role of species other than badgers in the epidemiology of bTB. Cultures were prepared from 10397 samples (primarily faeces, urine and tracheal aspirates). One of the 1307 bank voles (Clethrionomys glareolus) live-sampled, and three of the 43 badgers (Meles meles), yielded positive isolates of Mycobacterium bovis. This is the first time the bacterium has been isolated from the bank vole. The strain type was the same as that found in cattle and badgers on the same farm. However, our work indicates that the mean prevalence of infectious individuals among common farmland wildlife is extremely low (the upper 95% confidence interval is < or =2.0 for all of the abundant species). Mathematical models illustrate that it is highly unlikely the disease could be maintained at such low levels. Our results suggest that these animals are relatively unimportant as reservoirs of bTB, having insufficient within-species (or within-group) transmission to sustain the infection, though occasional spill-overs from cattle or badgers may occur.     

Mycobacterium tuberculosis Complex and HIV Co-Infection among Extrapulmonary Tuberculosis Suspected Cases at the University of Gondar Hospital,Northwestern Ethiopia

Background

Extrapulmonary Tuberculosis (EPTB) and Human Immunodeficiency Virus (HIV) infection are interrelated as a result of immune depression. The aim of this study was to determine the prevalence of Mycobacterium tuberculosis complex isolates and the burden of HIV co-infection among EPTB suspected patients.

Method

An institution based cross-sectional study was conducted among EPTB suspected patients at the University of Gondar Hospital. Socio-demographic characteristics and other clinical data were collected using a pretested questionnaire. GeneXpert MTB/RIF assay was performed to diagnosis Mycobacterium tuberculosis complex and Rifampicin resistance. All samples were also investigated by cytology and culture. The HIV statuses of all patients were screened initially by KHB, and all positive cases were further re-tested by STAT-pack. Data was analyzed using SPSS version 20 computer software and a P-value of < 0.05 was taken as statistically significant.

Results

A total of 141 extrapulmonary suspected patients were enrolled in this study. The overall prevalence of culture confirmed extrapulmonary tuberculosis infection was 29.8%, but the GeneXpert result showed a 26.2% prevalence of Mycobacterium tuberculosis complex infection. The 78.4% prevalence of extrapulmonary tuberculosis infection was found to be higher among the adult population. The prevalence of HIV infection among EPTB suspected patients was 14.1%, while it was 32.4% among GeneXpert-confirmed extrapulmonary TB cases (12/37). Tuberculosis lymphadenitis was the predominant (78.4%) type of EPTB infection followed by tuberculosis cold abscess (10.7%). Adult hood, previous history of contact with known pulmonary tuberculosis patients, and HIV co-infection showed a statistically significant association with extrapulmonary tuberculosis infection (P<0.013).

Conclusion

The prevalence of culture confirmed-EPTB infection was high, and a higher EPTB-HIV co-infection was also observed.     

Crystal Structures of the Kinase Domain of the Sulfate-Activating Complex in Mycobacterium tuberculosis

In Mycobacterium tuberculosis the sulfate activating complex provides a key branching point in sulfate assimilation. The complex consists of two polypeptide chains, CysD and CysN. CysD is an ATP sulfurylase that, with the energy provided by the GTPase activity of CysN, forms adenosine-5’-phosphosulfate (APS) which can then enter the reductive branch of sulfate assimilation leading to the biosynthesis of cysteine. The CysN polypeptide chain also contains an APS kinase domain (CysC) that phosphorylates APS leading to 3’-phosphoadenosine-5’-phosphosulfate, the sulfate donor in the synthesis of sulfolipids. We have determined the crystal structures of CysC from M. tuberculosis as a binary complex with ADP, and as ternary complexes with ADP and APS and the ATP mimic AMP-PNP and APS, respectively, to resolutions of 1.5 Å, 2.1 Å and 1.7 Å, respectively. CysC shows the typical APS kinase fold, and the structures provide comprehensive views of the catalytic machinery, conserved in this enzyme family. Comparison to the structure of the human homolog show highly conserved APS and ATP binding sites, questioning the feasibility of the design of specific inhibitors of mycobacterial CysC. Residue Cys556 is part of the flexible lid region that closes off the active site upon substrate binding. Mutational analysis revealed this residue as one of the determinants controlling lid closure and hence binding of the nucleotide substrate.     

Mycobacterium tuberculosis CwsA Interacts with CrgA and Wag31, and the CrgA-CwsA Complex Is Involved in Peptidoglycan Synthesis and Cell Shape Determination

Bacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein from Mycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog in M. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [¹⁴C]N-acetylglucosamine cell wall precursor. The M. smegmatis MSMEG_0023 crgA double mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA for cell wall synthesis and cell shape protein A, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.     

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France

We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.     

Global Effects of Inactivation of the Pyruvate Kinase Gene in the Mycobacterium tuberculosis Complex

To better understand the global effects of “natural” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis ΔpykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the ΔpykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in β-oxidation were increased in the M. tuberculosis ΔpykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress—AhpC, KatG, and SodA—showed increased synthesis in the ΔpykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the ΔpykA knockout, explaining why an M. tuberculosis ΔpykA Δald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has powerful, global effects on proteins associated with central metabolism.Comparison of the genome sequences of Mycobacterium bovis and Mycobacterium tuberculosis revealed >99.95% identity at the nucleotide level; however, these pathogens differ in terms of host tropism, phenotype, and virulence (16). Eleven regions of difference (RD) were observed in the M. bovis genome (2 to 12.7 kb) compared to M. tuberculosis, while one region deleted from M. tuberculosis was present in M. bovis (5, 16). In addition to the RDs, there are over 2,400 single nucleotide polymorphisms (SNPs) between M. tuberculosis and M. bovis (16). Some SNPs cosegregate with regions of deletions or other genetic markers (5); one such SNP is in the pykA gene, which cosegregates with the RD9 deletion. This SNP results in an inactive pyruvate kinase (PykA) being produced due to a Glu220Asp mutation (20). Glu220 is in the active site of the enzyme (21, 24), and its substitution results in complete loss of the enzyme activity in M. bovis (20). Thus, the pykA SNP explains one of the classic distinctions between M. bovis and M. tuberculosis, namely, the requirement for pyruvate. Neither glycerol, the preferred carbon source for isolation of tubercle bacilli, nor glucose support the growth of M. bovis when they are not supplemented with pyruvate (38), due to the inactive pyruvate kinase.On the routinely used Middlebrook 7H11 agar, containing glycerol and oleate, M. bovis shows dysgonic colony morphology, whereas M. tuberculosis, in contrast, shows eugonic colony morphology with abundant growth. Complementation of M. bovis with the active pykA gene from M. tuberculosis restored the eugonic phenotype. Thus, loss of PykA activity commits M. bovis to using nonglycolytic substrates as carbon sources, such as lipids. This in itself is of biological significance since human M. tuberculosis switches to this kind of metabolism in experimentally infected animals or in macrophages (34, 35, 39). However, even with oleate (a lipid) as a sole carbon source which allows both species to grow, there was still a difference in colony morphology (20). This led us to consider that loss of the pykA gene had wider effects since PykA is not needed for energy production on oleate and has no role in gluconeogenesis (Fig. ​(Fig.1).1). Thus, we hypothesize that the loss of the pykA gene has global effects over and above the predicted effect of determining whether or not growth can take place on glycerol. To examine our hypothesis, we created a pykA mutant of M. tuberculosis to investigate the effect of pykA deletion by using isogenic strains. This builds upon our previous study in which we had complemented M. bovis with the (active) M. tuberculosis pykA gene (20). We also created a mutant in alanine dehydrogenase (H37Rv Δald) and a H37Rv Δald ΔpykA double mutant since M. bovis naturally lacks active ald and pykA genes (16). The global effects of these knockout mutations were then examined by their on growth on a range of carbon sources and on protein expression during growth on pyruvate, a gluconeogenic carbon source. A proteomic approach was chosen since it would reveal changes in all proteins, for example, regulatory proteins, enzymes, and stress proteins; key proteins, or effects of changes in their levels, could then be assayed for directly. These approaches revealed the major metabolic consequences resulting from pykA inactivation.Open in a separate windowFIG. 1.Pathways of carbon metabolism possible in strains with or without pyruvate kinase (PykA). Boxes denote substrates and/or products where arrows are used to denote pathways. Arrows to and from boxes are pathways; other arrows show reactions catalyzed by a single enzyme. Substrates are in text with serifs; pathways and enzymes in plain text. Colored arrows are used to denote glycolysis or gluconeogenesis in red, the tricarboxylic acid cycle in blue, and the glyoxylate cycle in magenta.     

新疆喀什维吾尔族结核病患者结核分枝杆菌5个VNTRs基因分型研究

目的:应用多位点数目可变串联重复序列分析(multiple loci VNTR analysis,MLVA)技术,对新疆喀什地区维吾尔族结核病患者结核分枝杆菌临床分离株进行基因分型,探讨5个数目可变串联重复序列(VNTR)基因型种类及其分布。方法:收集结核分枝杆菌,采用PCR和琼脂糖凝胶电泳技术,结合BioNumerics5.0软件,对其5个VNTR位点进行结果分析。结果:分离出58株结核分枝杆菌,分为4个基因群21个基因型,分别为Ⅰ群占19.1%,含7个基因型;Ⅱ群占3.4%,含2个基因型;Ⅲ群占67.2%,含9个基因型;Ⅳ群占10.3%,含5个基因型。结论:新疆喀什地区维吾尔族结核病患者的结核分枝杆菌存在明显的基因多态性,且存在主要流行菌群。