Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)–amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH₂ (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.     

Identification and comparative analysis of a genomic island in Mycobacterium avium subsp. hominissuis

Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacterium causing opportunistic infections. The objective of this study was to identify flexible genome regions in MAH isolated from different sources. By comparing five complete and draft MAH genomes we identified a genomic island conferring additional flexibility to the MAH genomes. The island was absent in one of the five strains and had sizes between 16.37 and 84.85 kb in the four other strains. The genes present in the islands differed among strains and included phage- and plasmid-derived genes, integrase genes, hypothetical genes, and virulence-associated genes like mmpL or mce genes.     

副结核分枝杆菌感染的免疫应答与检测方法研究进展北大核心CSCD

副结核分枝杆菌常引起感染牛的产奶量下降、持续性消瘦、顽固性下痢甚至死亡,给畜牧业带来了巨大的经济损失。牛通常在幼年期经口感染该菌并具有一个较长的亚临床期,后期才表现出临床症状,感染前期以细胞免疫为主并伴随着间歇排菌,经过一个2到5年的亚临床期后体液免疫应答增强,同时排菌量明显增加。目前对牛副结核病常用的检测方法有病原学检测方法,基于细胞免疫反应和基于体液免疫反应的检测方法。由于不同方法的反应原理不同,加之副结核分枝杆菌感染动物的特定免疫应答规律,在某一时间内各方法之间敏感性差异较大。本文简要阐述了牛副结核分枝杆菌的传播途径以及牛感染后的免疫应答特点,对牛副结核病的常见诊断方法进行了综述。     

Role of the mitogen-activated protein kinase pathway in the differential response of bovine monocytes to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

We compared the kinetics of activation and antimicrobial activities of MAPK-p38 and MAPK-ERK in bovine monocytes infected with Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium avium subsp. avium (Maa). Monocytes were incubated with MAP or Maa organisms with or without a specific inhibitor of the MAPK-p38 pathway (SB203580), and MAPK phosphorylation and antimicrobial functions of monocytes were evaluated. At early time points MAPK-p38 phosphorylation was greater in MAP-infected bovine monocytes than in Maa-infected monocytes. At later time points MAPK-p38 phosphorylation by both organisms was similar. MAPKp38 phosphorylation in MAP-infected monocytes was similar to negative control cells, whereas in Maa-infected this activation remained greater than negative control cells. Increase phosphorylation MAPK-ERK was similar at all time points for both organisms. Bovine monocytes had minimal capacity to kill MAP organisms, to acidify MAP-containing phagosomes, or to form phagolysosome. Alternatively, bovine monocytes were able to kill Maa organisms. Addition of SB203580 to monocyte cultures increased phagosome acidification, phagolysosome formation, and killing of MAP and Maa organisms. Taken together these data indicate that early transient activation of MAPK-p38 in bovine mononuclear phagocytes by MAP organisms may be a key mechanism involved in the capacity of MAP to survive in bovine monocytes.     

Endometrial inflammation and abnormal expression of extracellular matrix proteins induced by Mycoplasma bovis in dairy cows

Mycoplasma bovis infection can cause endometrial inflammation leading to infertility and involuntary culling in dairy cows. Because extracellular matrix (ECM) proteins affect the adherence of mycoplasma to eukaryotic cell surface, they may play a role in the pathogenesis of the bacteria. The objective of the present study was to evaluate the endometrial inflammatory response and ECM protein expression induced by M bovis. Endometrial concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and mRNA and protein expression of collagen IV (CL-IV), fibronectin (FN), and laminin (LN) were evaluated 10, 20, and 30 days after M bovis intrauterine infusion in breed cows 18 days postpartum. The presence of the bacteria in the uterus was detected by nested polymerase chain reaction and denaturing gradient gel electrophoresis. Endometrial TNF-α, IL-1β, and IL-6 concentrations in the treatment group were greater (P < 0.05) than in the positive and negative control groups 20 and 30 days after infusion. Endometrial CL-IV, FN, and LN mRNA and protein expression increased (P < 0.01) 20 days after infusion in all groups. However, the increase was more pronounced in the treatment group and reactive expressions were greater (P < 0.05) than in the positive and negative control groups 10, 20, and 30 days after infusion. In conclusion, M bovis triggered endometrial inflammatory response and increased CL-IV, FN, and LN mRNA and protein expression. The abnormal expression of ECM these proteins may promote the pathogenic effects of M bovis that lead to endometrial tissue damage and infertility.     

Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h

The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease.     

High resolution melting PCR to differentiate Mycobacterium avium subsp. paratuberculosis"cattle type" and "sheep type"

In this study, we describe a novel HRM-PCR assay that clearly differentiates the two main types of Mycobacterium avium subsp. paratuberculosis (cattle and sheep) based on the polymorphic variation of a previously described tandem repeat. This modern genotyping technique has several advantages over alternative methods, including cost, ease of use and rapidity.     

Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness

Background

Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate intracellular pathogen that infects many ruminant species. The acquisition of foreign genes via horizontal gene transfer has been postulated to contribute to its pathogenesis, as these genetic elements are absent from its putative ancestor, M. avium subsp. hominissuis (MAH), an environmental organism with lesser pathogenicity. In this study, high-throughput sequencing of MAP transposon libraries were analyzed to qualitatively and quantitatively determine the contribution of individual genes to bacterial survival during infection.

Results

Out of 52384 TA dinucleotides present in the MAP K-10 genome, 12607 had a MycoMarT7 transposon in the input pool, interrupting 2443 of the 4350 genes in the MAP genome (56%). Of 96 genes situated in MAP-specific genomic islands, 82 were disrupted in the input pool, indicating that MAP-specific genomic regions are dispensable for in vitro growth (odds ratio = 0.21). Following 5 independent in vivo infections with this pool of mutants, the correlation between output pools was high for 4 of 5 (R = 0.49 to 0.61) enabling us to define genes whose disruption reproducibly reduced bacterial fitness in vivo. At three different thresholds for reduced fitness in vivo, MAP-specific genes were over-represented in the list of predicted essential genes. We also identified additional genes that were severely depleted after infection, and several of them have orthologues that are essential genes in M. tuberculosis.

Conclusions

This work indicates that the genetic elements required for the in vivo survival of MAP represent a combination of conserved mycobacterial virulence genes and MAP-specific genes acquired via horizontal gene transfer. In addition, the in vitro and in vivo essential genes identified in this study may be further characterized to offer a better understanding of MAP pathogenesis, and potentially contribute to the discovery of novel therapeutic and vaccine targets.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-415) contains supplementary material, which is available to authorized users.     

The evolutionary landscape of the Mycobacterium tuberculosis genome

Mycobacterium tuberculosis is one of the most deadly human pathogens. The major mechanism for the adaptations of M. tuberculosis is nucleotide substitution. Previous studies have relied on the nonsynonymous-to-synonymous substitution rate (d_N/d_S) ratio as a measurement of selective constraint based on the assumed selective neutrality of synonymous substitutions. However, this assumption has been shown to be untrue in many cases. In this study, we used the substitution rate in intergenic regions (d_i) of the M. tuberculosis genome as the neutral reference, and conducted a genome-wide profiling for d_i, d_S, and the rate of insertions/deletions (indel rate) as compared with the genome of M. canettii using a 50 kb sliding window. We demonstrate significant variations in all of the three evolutionary measurements across the M. tuberculosis genome, even for regions in close vicinity. Furthermore, we identified a total of 233 genes with their d_S deviating significantly from d_i within the same window. Interestingly, d_S also varies significantly in some of the windows, indicating drastic changes in mutation rate and/or selection pressure within relatively short distances in the M. tuberculosis genome. Importantly, our results indicate that selection on synonymous substitutions is common in the M. tuberculosis genome. Therefore, the d_N/d_S ratio test must be applied carefully for measuring selection pressure on M. tuberculosis genes.     

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10⁴-10^− 1) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10² and 10³ MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1 MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6 weeks by real-time PCR.     

Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex: a basis for pathogenicity

Background

A human isolate of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis 43525) was sequenced and compared genomically to other mycobacterial pathogens. M. paratuberculosis 43525 was recently isolated from a patient with ulcerative colitis and belongs to the M. avium complex, a group known to infect both humans and animals. While M. paratuberculosis is a known pathogen of livestock, there are only 20 human isolates from the last 20 years, therefore we took the opportunity to perform a whole genome comparison between human and animal mycobacterial pathogens. We also compared virulence determinants such as the mycobactin cluster, PE/PPE genes and mammalian cell entry (mce) operons between MAC subspecies that infect animals and those that infect humans. M. tuberculosis was also included in these analyses given its predominant role as a human pathogen.

Results

This genome comparison showed the PE/PPE profile of M. paratuberculosis 43525 to be largely the same as other M. paratuberculosis isolates, except that it had one PPE and one PE_PGRS protein that are only present in human MAC strains and M. tuberculosis. PE/PPE proteins that were unique to M. paratuberculosis 43525, M. avium subsp. hominissuis and a caprine M. paratuberculosis isolate, were also identified. In addition, the mycobactin cluster differed between human and animal isolates and a unique mce operon flanked by two mycobactin genes, mbtA and mbtJ, was identified in all available M. paratuberculosis genomes.

Conclusions

Despite the whole genome comparison placing M. paratuberculosis 43525 as closely related to bovine M. paratuberculosis, key virulence factors were similar to human mycobacterial pathogens. This study highlights key factors of mycobacterial pathogenesis in humans and forms the basis for future functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1889-2) contains supplementary material, which is available to authorized users.     

A duplex real-time PCR assay for detection of mutT4 and mutT2 mutations in Mycobacterium tuberculosis of Beijing genotype

To determine the polymorphism of mutT genes of Mycobacterium tuberculosis of Beijing genotype, we developed a duplex real-time PCR assay based on hybridization probes for the Roche LightCycler instrument. The assay rapidly detects mutations at codons 48 and 58 of genes mutT4 and at mutT2, respectively.     

Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system

Mycobacterium tuberculosis reduces nitrate very strongly as compared to Mycobacterium bovis and M. bovis BCG. Nitrate reductase, in conjunction with niacin accumulation, constitutes one of the major biochemical tests used in clinical microbiology laboratories to differentiate M. tuberculosis from other members of the M. tuberculosis complex, as well as nontuberculous Mycobacteria. Determination of nitrate reductase activity is currently performed using cultures grown on solid media with a slow detection time and the need for large quantities of bacilli, as otherwise the test is not reliable. Hereby, we propose a nitrate reduction test coupled to Bactec MGIT960 system as a simple, rapid and economic method with a total gain of time of about 3 to 4 weeks over the conventional solid medium. In our study, almost all the M. tuberculosis and Mycobacterium canettii strains gave a strongly positive nitrate reductase result within 1 day of positive detection by the MGIT960 system. In contrast, M. bovis, M. bovis BCG and M. africanum strains remained negative even after 14 days of incubation. The possibility to detect nitrate reductase within 1 to 3 days of a positive culture using MGIT960 opens new perspectives with the possibility of confirming M. tuberculosis — starting directly from pathological specimens.     

Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), evades the antimicrobial defenses of the host and survives within the infected individual through a complex set of strategies. These include active prevention of host cellular killing processes as well as overwhelming adaptive gene expression. In the past decade, we have gained an increased understanding of how mycobacteria not only have the ability to adapt to a changing host environment but also actively interfere with the signaling machinery within the host cell to counteract or inhibit parts of the killing apparatus employed by the macrophage. Mtb is able to sense its environment via a set of phospho-signaling proteins which mediate its response and interaction with the host in a coordinated manner. In this review, we summarize the current knowledge about selected Mtb serine, threonine, and tyrosine kinase and phosphatase signaling proteins, focusing on the protein kinases, PknG and PtkA, and the protein phosphatase, PtpA.     

The telocentric tandem repeat at the p-arm is not conserved in Mus musculus subspecies

Mouse chromosomes, with the exception of the Y chromosome, are telocentric. The telomere at the p-arm is separated from the centromere by the tL1 sequence and TLC tandem repeats. A previous report showed that the TLC array was also conserved in other strains of the subgenus Mus. These results suggest that the TLC arrays promote the stable evolutionary maintenance of a telocentric karyotype in the subgenus Mus. In this study, we investigated the degree of conservation of TLC arrays among a variety of wild-derived inbred strains, all of which are descendants of wild mice captured in several areas of the world. Genomic PCR analysis indicates that the sequential order of telomere-tL1 is highly conserved in all strains, whereas tL1-TLC is not. Next, Southern blot analysis of DNAs isolated from a panel of mouse subspecies showed both Mus musculus domesticus and Mus musculus castaneus subspecies possess TLC arrays. Unexpectedly, this repeat appears to be lost in almost all Mus musculus musculus and Mus musculus molossinus subspecies, which show a clear geographic divide. These results indicate that either other unknown sequences were replaced by the TLC repeat or almost all M. m. musculus and M. m. molossinus subspecies do not have any sequence between the telomere and minor satellites. Our observation suggests that the TLC array might be evolutionarily unstable and not essential for murine chromosomal conformation. This is the first example of the subspecies-specific large genome alterations in mice.     

Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis

Genetic research in molecular laboratories relies heavily on directed mutagenesis and gene deletion techniques. In mycobacteria, however, genetic analysis is often hindered by difficulties in the preparation of deletion mutants. Indeed, in comparison to the allelic exchange systems available for the study of other common model organisms, such as Saccharomyces cerevisiae and Escherichia coli, mycobacterial gene disruption systems suffer from low mutant isolation success rates, mostly due to inefficient homologous recombination and a high degree of non-specific recombination. Here, we present a gene deletion system that combines efficient homologous recombination with advanced screening of mutants. This novel methodology allows for gene disruption in three consecutive steps. The first step relies on the use of phage Che9c recombineering proteins for directed insertion into the chromosome of a linear DNA fragment that encodes GFP and confers hygromycin resistance. In the second step, GFP positive and hygromycin resistant colonies are selected, and in the last step, the gfp-hyg cassette is excised from the chromosome, thus resulting in the formation of an unmarked deletion. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the prcSBA operon of Mycobacterium smegmatis.     

Comparative analysis of iron regulated genes in mycobacteria

Iron dependent regulator, IdeR, regulates the expression of genes in response to intracellular iron levels in M. tuberculosis. Orthologs of IdeR are present in all the sequenced genomes of mycobacteria. We have used a computational approach to identify conserved IdeR regulated genes across the mycobacteria and the genes that are specific to each of the mycobacteria. Novel iron regulated genes that code for a predicted 4-hydroxy benzoyl coA hydrolase (Rv1847) and a protease dependent antibiotic regulatory system (Rv1846c, Rv0185c) are conserved across the mycobacteria. Although Mycobacterium natural-resistance-associated macrophage protein (Mramp) is present in all mycobacteria, it is, as predicted, an iron-regulated gene in only one species, M. avium subsp. paratuberculosis. We also observed an additional iron-regulated exochelin biosynthetic operon, which is present only in non-pathogenic Mycobacterium, M. smegmatis.