Characterization of Mce4A protein of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: role in invasion and survival

Background  

The mce4 operon is one of the four homologues of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. The mce4A (Rv3499c) gene within this operon is homologous to mce1A (Rv0169), that has a role in host cell invasion by M. tuberculosis. Our earlier reports show that mce4 operon is expressed during the stationary phase of growth of the bacillus in culture and during the course of infection in mammalian hosts. M. tuberculosis carrying mutation in mce4 operon shows growth defect and reduced survival in infected mice. However, the intracellular localization of Mce4A protein and its direct role in cell entry or survival of the bacillus has not been demonstrated so far.     

Expression,purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901

Aims: The main aims of this study were to clone and express flagellin flaA gene from Vibrio alginolyticus strain HY9901, also to prepare mouse anti‐FlaA polyclonal antibody for future pathogen or vaccine study. Methods and Results: The full‐length flaA gene was amplified by PCR with designed primers. The open reading frame of flaA gene contains 1131 bp, and its putative protein consists of 376 amino acid residues. Alignment analysis indicated that the FlaA protein was highly conserved. SDS–PAGE indicated that the FlaA protein was successfully expressed in Escherichia coli BL21 (DE3). Then, the recombinant FlaA protein was purified by affinity chromatography, and the mouse anti‐FlaA serum was produced. The expression of flaA gene was verified by various immunological methods, including western blotting, enzyme‐linked immunosorbent assay (ELISA) and immunogold electron microscopy (IEM). Conclusions: Flagellin flaA gene was cloned and identified from V. alginolyticus HY9901, the recombinant FlaA protein was expressed and purified, and high‐titre FlaA protein‐specific antibody was produced. Western blot analysis revealed that the prepared antiserum not only specifically react to FlaA fusion protein, but also to natural FlaA protein of V. alginolyticus. The expressed FlaA protein was demonstrated, for the first time, as the component of flagella from V. alginolyticus by IEM. Significance and Impact of the Study: This study may offer important insights into the pathogenesis of V. alginolyticus, provide a base for further studies on the diagnosis and evaluation that whether the FlaA protein could be used as an effective vaccine candidate against infection by V. alginolyticus and other Vibrio species. Additionally, the purified FlaA protein and polyclonal antibody can be used for further functional and structural studies.     

Cloning,expression and characterization of attachment‐invasion locus protein (Ail) of Yersinia enterocolitica and its utilization in rapid detection by immunoassays

Aims: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment‐invasion locus (Ail) protein. Methods and Results: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l^?1 final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. Conclusion: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. Significance and Impact of the Study: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.     

Immunohistochemical staining of macrophages in the skin lesions of leprosy: the role of antibody to mycobacteria in human serum and various polyclonal immune rabbit antisera

Summary Immunohistochemical staining of tuberculoid and lepromatous leprosy skin lesions was performed using various rabbit antisera. Macrophages in both stained with serum containing antibodies against lysozyme and alpha-1-antitrypsin, while macrophages in lepromatous leprosy also reacted with other antibodies. An immunoglobulin fraction of positive serum stained following pepsin digestion, indicating that reactivity was not Fc dependent. Positive serum contained antibody againstMycobacterium butyricum, which caused macrophage staining, since affinity-purified antibody did not stain and absorption withM. butyricum removed staining. Staining was also produced by serum of subjects with leprosy or a positive tuberculin test. By immunoblotting, the anti-mycobacterial antibody was directed against surface components ofM. butyricum of molecular weights 20 000–70 000. Electron microscopy showedM. leprae in phagolysosomes of macrophages, while immunoelectron microscopy demonstrated labelling along bacterial cell membranes. Therefore, macrophages in lepromatous leprosy skin lesions stain because they containM. leprae, which reacts with antibody to eitherM. leprae, M. tuberculosis or atypical mycobacteria in human serum and with antibody toM. butyricum in serum from rabbits immunized with various antigens and Freund's complete adjuvant. These results indicate that immunohistochemical studies on leprosy are misleading if performed using intact polyclonal immune sera rather than affinity purified or monoclonal antibodies.     

Combining comparative genomic analysis with machine learning reveals some promising diagnostic markers to identify five common pathogenic non-tuberculous mycobacteria

Non-tuberculous mycobacteria (NTM) can cause various respiratory diseases and even death in severe cases, and its incidence has increased rapidly worldwide. To date, it’s difficult to use routine diagnostic methods and strain identification to precisely diagnose various types of NTM infections. We combined systematic comparative genomics with machine learning to select new diagnostic markers for precisely identifying five common pathogenic NTMs (Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium chelonae, Mycobacterium abscessus). A panel including six genes and two SNPs (nikA, benM, codA, pfkA2, mpr, yjcH, rrl C2638T, rrl A1173G) was selected to simultaneously identify the five NTMs with high accuracy (> 90%). Notably, the panel only containing the six genes also showed a good classification effect (accuracy > 90%). Additionally, the two panels could precisely differentiate the five NTMs from M. tuberculosis (accuracy > 99%). We also revealed some new marker genes/SNPs/combinations to accurately discriminate any one of the five NTMs separately, which provided the possibility to diagnose one certain NTM infection precisely. Our research not only reveals novel promising diagnostic markers to promote the development of precision diagnosis in NTM infectious, but also provides an insight into precisely identifying various genetically close pathogens through comparative genomics and machine learning.     

Molecular mechanism of interaction of Mycobacterium tuberculosis with host macrophages under high glucose conditions

Mycobacterium tuberculosis has the potential to escape various cellular defense mechanisms for its survival which include various oxidative stress responses, inhibition of phagosome-lysosomes fusion and alterations in cell death mechanisms of host macrophages that are crucial for its infectivity and dissemination. Diabetic patients are more susceptible to developing tuberculosis because of impairement of innate immunity and prevailing higher glucose levels. Our earlier observations have demonstrated alterations in the protein profile of M. tuberculosis exposed to concurrent high glucose and tuberculosis conditions suggesting a crosstalk between host and pathogen under high glucose conditions. Since high glucose environment plays crucial role in the interaction of mycobacterium with host macrophages which provide a niche for the survival of M. tuberculosis, it is important to understand various interactive mechanisms under such conditions. Initial phagocytosis and containment of M. tuberculosis by macrophages, mode of macrophage cell death, respiratory burst responses, Mycobacterium and lysosomal co-localization were studied in M. tuberculosis H37Rv infected cells in the presence of varied concentrations of glucose in order to mimic diabetes like conditions. It was observed that initial attachment, phagocytosis and later containment were less effective under high glucose conditions in comparison to normal glucose. Mycobacterium infected cells showed more necrosis than apoptosis as cell death mechanism during the course of infection under high glucose concentrations. Co-localization and respiratory burst assay also indicated evasion strategies adopted by M. tuberculosis under such conditions. This study by using THP1 macrophage model of tuberculosis and high glucose conditions showed immune evasion strategies adapted during co-pathogenesis of tuberculosis and diabetes.     

Leishmania infection inhibits macrophage motility by altering F‐actin dynamics and the expression of adhesion complex proteins

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self‐healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis‐infected macrophages also show reduced directional migration in response to the chemokine MCP‐1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F‐actin turnover frequency in L. amazonensis‐infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions.     

The <Emphasis Type="Italic">Mycobacterium marinum mel2</Emphasis> locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species

Background  

Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood.     

Identification of Mycobacterium species from apparently healthy freshwater aquarium fish using partial sequencing and PCR‐RFLP analysis of heat shock protein (hsp65) gene

The present study analysed the incidence of mycobacteria in apparently healthy looking freshwater aquarium fish in Uttar Pradesh (State), India. Sixty fish belonging to eight different species were collected from six aquarium shops in different cities and processed for isolation of Mycobacterium species. Using the initial protocol of decontamination of tissue homogenates (with 1N HCl & 2N NaOH) and incubation at 30°C for 2 months, Mycobacterium sp. was isolated from 25% of the fish. The isolates were identified by standard biochemical tests. A 441 bp fragment of the hsp65 gene was amplified and digested by two fastdigest restriction enzymes, BstEII and HaeIII. Digested products were analysed using agarose gel electrophoresis. Sequencing of amplified fragments of the hsp65 gene was also performed. Isolates were identified as: five isolates of M. abscessus, three M. gordonae, two M. fortuitum, two M. conceptionense, two M. parascrofulaceum, and one isolate of M. senegalense. Mycobacterial incidence in apparently healthy looking freshwater aquarium fish is dreadful and the study is relevant because of the mycobacterial diversity related to aquarium fish and its zoonotic importance. All Mycobacterium species isolated in this study are well known pathogens in humans as well as fish.     

Mycobacteria employ two different mechanisms to cross the blood–brain barrier

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood–brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX‐1 secretion system, which extends the role of ESX‐1 secretion beyond the macrophage infection cycle.     

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.     

Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains

Background  

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system.     

Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern,phagosome maturation,and escape to the cytosol

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication‐permissive compartment termed the Mycobacterium‐containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid‐borne genes. Fluorescence microscopy of M. marinum‐infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol‐3‐phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V‐ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.     

Macrophage surface glycoproteins binding to galectin-3 (Mac-2-antigen)

Galectin-3 (formerly called Mac-2 antigen) is a ∼30 kDa carbohydrate-binding protein expressed on the surface of inflammatory macrophages and several macrophage cell lines. We have purified from lysates of the murine macrophage cell line WEHI-3 glycoproteins that bind to a galectin-3 affinity column. Several of these receptors are labelled after biotinylation of intact cells showing their location at the cell surface. N-terminal aminoacid sequencing of intact galectin-3-binding glycoproteins isolated from preparative SDS-gels or of chemically derived fragments showed several homologies with known proteins and identification was confirmed by immunoprecipitation with specific antibodies. The glycoproteins were shown to be: the α-subunit(CD11b) of the CD11b/CD18 integrin(Mac-1 antigen); the lysosomal membrane glycoproteins LAMPs 1 and 2 which are known in part to be expressed at cell surfaces; the Mac-3 antigen, a mouse macrophage differentiation antigen defined by the M3/84 monoclonal antibody and related immunochemically to LAMP-2; the heavy chain of CD98, a 125 kDa heterodimeric glycoprotein identified by the 4F2/RL388 monoclonal antibodies respectively on human and mouse monocytes/macrophages and on activated T cells. Further studies showed that CD11b/CD18, CD98 and Mac-3 are major surface receptors for galectin-3 on murine peritoneal macrophages elicited by thioglycollate. Abbreviations: PBS, phosphate buffered saline; CNBR, cyanogen bromide; PMSF, phenyl methyl sulphonyl fluoride This revised version was published online in November 2006 with corrections to the Cover Date.     

Binding to type I collagen is essential for the infectivity of Vibrio parahaemolyticus to host cells

Vibrio parahaemolyticus is a globally present marine bacterium that often leads to acute gastroenteritis. Two type III secretion systems (T3SSs), T3SS1 and T3SS2, are important for host infection. Type I collagen is a component of the extracellular matrix and is abundant in the small intestine. However, whether type I collagen serves as the cellular receptor for V. parahaemolyticus infection of host cells remains enigmatic. In this study, we discovered that type I collagen is not only important for the attachment of V. parahaemolyticus to host cells but is also involved in T3SS1‐dependent cytotoxicity. In addition, 2 virulence factors, MAM7 and VpadF enable V. parahaemolyticus to interact with type I collagen and mediate T3SS2‐dependent host cell invasion. Type I collagen, the collagen receptor α1 integrin, and its downstream factor phosphatidylinositol 3‐kinase (PI3K) are responsible for V. parahaemolyticus invasion of host cells. Further biochemical studies revealed that VpadF mainly relies on the C‐terminal region for type I collagen binding and MAM7 relies on mce domains to bind to type I collagen. As MAM7 and/or VpadF homologues are widely distributed in the genus Vibrio, we propose that Vibrios have evolved a unique strategy to infect host cells by binding to type I collagen.     

Sulfolipid accumulation in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> disrupted in the <Emphasis Type="Italic">mce2</Emphasis> operon

Mycobacterium tuberculosis, the causative agent of tuberculosis, has a lipid-rich cell wall that serves as an effective barrier against drugs and toxic host cell products, which may contribute to the organism’s persistence in a host. M. tuberculosis contains four homologous operons called nice (mce1–4) that encode putative ABC transporters involved in lipid importation across the cell wall. Here, we analyzed the lipid composition of M. tuberculosis disrupted in the mce2 operon. High resolution mass spectrometric and thin layer chromatographic analyses of the mutant’s cell wall lipid extracts showed accumulation of SL-1 and SL₁₂₇₈ molecules. Radiographic quantitative analysis and densitometry revealed 2.9, 3.9 and 9.8-fold greater amount of [³⁵S] SL-1 in the mce2 operon mutant compared to the wild type M. tuberculosis during the early/mid logarithmic, late logarithmic and stationary phase of growth in liquid broth, respectively. The amount of [³⁵S] SL₁₂₇₈ in the mutant also increased progressively over the same growth phases. The expression of the mce2 operon genes in the wild type strain progressively increased from the logarithmic to the stationary phase of bacterial growth in vitro, which inversely correlated with the proportion of radiolabel incorporation into SL-1 and SL₁₂₇₈ at these phases. Since the mce2 operon is regulated in wild type M. tuberculosis, its cell wall may undergo changes in SL-1 and SL₁₂₇₈ contents during a natural course of infection and this may serve as an important adaptive strategy for M. tuberculosis to maintain persistence in a host.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.