WASH‐driven actin polymerization is required for efficient mycobacterial phagosome maturation arrest

Pathogenic mycobacteria survive in phagocytic host cells primarily as a result of their ability to prevent fusion of their vacuole with lysosomes, thereby avoiding a bactericidal environment. The molecular mechanisms to establish and maintain this replication compartment are not well understood. By combining molecular and microscopical approaches we show here that after phagocytosis the actin nucleation‐promoting factor WASH associates and generates F‐actin on the mycobacterial vacuole. Disruption of WASH or depolymerization of F‐actin leads to the accumulation of the proton‐pumping V‐ATPase around the mycobacterial vacuole, its acidification and reduces the viability of intracellular mycobacteria. This effect is observed for M. marinum in the model phagocyte Dictyostelium but also for M. marinum and M. tuberculosis in mammalian phagocytes. This demonstrates an evolutionarily conserved mechanism by which pathogenic mycobacteria subvert the actin‐polymerization activity of WASH to prevent phagosome acidification and maturation, as a prerequisite to generate and maintain a replicative niche.     

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.     

Synthetic thiosemicarbazones as a new class of Mycobacterium tuberculosis protein tyrosine phosphatase A inhibitors

Mycobacterium tuberculosis secretes two protein tyrosine phosphatases as virulence factors, PtpA and PtpB. Inhibition studies of these enzymes have shown significant attenuation of the M. tuberculosis growth in vivo. As PtpA mediates many effects on the regulation of host signaling ensuring the intracellular survival of the bacterium we report, for the first time, thiosemicarbazones as potential novel class of PtpA inhibitors. Several compounds were synthesized and biologically evaluated, revealing interesting results. Enzyme kinetic assays showed that compounds 5, 9 and 18 are non-competitive inhibitors of PtpA, with K_i values ranging from 1.2 to 5.6?µM. Modeling studies clarified the structure-activity relationships observed in vitro and indicated a possible allosteric binding site in PtpA structure. To the best of our knowledge, this is the first disclosure of potent non-competitive inhibitors of PtpA with great potential for future studies and development of analogues.     

Genotypic characteristics of a Mycobacterium sp. isolated from yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex in Japan

In Japan, a Mycobacterium marinum‐like mycobacterium was isolated from the yellowtail, Seriola quinqueradiata. The species was identified as M. marinum by a commercial mycobacterial DNA‐DNA hybridization kit. Nevertheless, PCR restriction analysis of the DNA of its RNA polymerase β‐subunit gene definitively showed that this Mycobacterium sp. was M. ulcerans. PCR analysis revealed the genotypic characteristics of M. ulcerans in the Mycobacterium sp., only the mup053 gene sequence being absent, as has been found previously in other piscine mycobacteria such as M. marinum strains DL240490 and DL045 and M. pseudoshottsii. With one exception, this Mycobacterium sp. and M. pseudoshottsii had identical 16S rRNA gene sequences, which is also probably true of M. marinum strains DL240490 and DL045. Similarly, according to comparisons of the 16S rRNA gene, ITS region, and hsp65 gene sequences, this Mycobacterium sp. is more closely related to M. pseudoshottsii than to M. ulcerans or M. marinum. A PCR product of approximately 2000 bp was amplified from region of difference 9 in the Mycobacterium sp. The nucleotide sequence revealed insertion of IS2404, the sequence of which is 1366 bp long. The novel single nucleotide polymorphisms identified in this region distinguished this Mycobacterium sp. from M. marinum strain DL240490 and M. pseudoshottsii. The present findings raise the possibility that these species have a common ancestor. Further studies are required to improve our understanding of the relationship between their geographical origin and genetic diversity.     

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes

Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium‐containing vacuole, possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential yet poorly soluble and available micronutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid‐bound mycobactin (MBT) and the water‐soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialised phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe‐MBT or Fe‐cMBT improved the growth of wild type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe‐cMBT promoted the growth of wild‐type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by coinfection with wild‐type bacteria. Our study identifies and characterises the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extra‐ and intracellular growth of the pathogen.     

Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P‐mediated autophagy‐independent pathway

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterised by a long‐lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3‐phosphate (PI3P)‐labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P‐labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3‐labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.     

Comparative Genome Analysis of Fish and Human Isolates of Mycobacterium marinum

Mycobacterium marinum is a major causative agent of mycobacteriosis in fish that has a broad range of hosts, including in human isolates. So far, genomic analyses have focused on the human isolate. Here, we compared the draft genome sequences of two strains of M. marinum isolated from fish (MB2 and Europe) with the M. marinum M isolated from humans. M. marinum MB2 and Europe have single, circular chromosomes of 6,134,389 and 6,029,340 bp, and average G + C contents of 65.7 and 65.5 %, respectively. A total of 5,464 coding DNA sequences were annotated in both M. marinum MB2 and Europe genome. Dot plot analyses showed that M. marinum MB2 and Europe were closer to M. marinum M when compared to three other Mycobacterium species. The insertion/deletion gene analysis showed that M. marinum MB2 and Europe contained 342 and 487 genes that were not found in M. marinum M, and lacked 625 and 776 genes found in M. marinum M, respectively. Most of the inserted and deleted genes were classified in the fatty acid, lipid, and isoprenoid subsystem and the virulence, disease, and defense subsystem. Therefore, these results provide insights into the genomic diversity associated with variable hosts and pathogens.     

Morphology and Phylogeny of Three Pleuronema Species (Ciliophora,Scuticociliatia) from Hangzhou Bay,China, with Description of Two New Species,P. binucleatum n. sp. and P. parawiackowskii n. sp.

The morphology and phylogeny of Pleuronema binucleatum n. sp., P. parawiackowskii n. sp., and P. marinum Dujardin 1841, collected from Hangzhou Bay estuary, China, were investigated using standard methods. Pleuronema binucleatum n. sp. can be identified by possessing about 90–120 × 35–50 μm cell size in vivo, reniform body outline, two macronuclei, six to eight preoral kineties, 32–41 somatic kineties, and posterior end of the anterior fragment of membranelle 2 (M2a) hook‐like. Pleuronema parawiackowskii n. sp. is characterized by the combination of the following characters: body size about 60–90 × 20–40 μm in vivo, elliptical in outline, four to eight preoral kineties, 20–29 somatic kineties, posterior portion of the M2a slightly curved but nonhooked, and single macronucleus sausage‐like. After comparison with other populations of P. marinum, it is suggested that many misidentifications exist in previous studies. And an improved diagnosis of P. marinum was supplied: cell about 95–180 μm long, elliptical in outline; 2–4 preoral kineties and 53–70 somatic kineties; both membranelle 1 and membranelle 3 three‐rowed; posterior end of the M2a straight; single contractile vacuole characteristically positioned near mid‐body. The small subunit rRNA genes of three forms were sequenced. Phylogenetic analyses indicate that the monophyly of the genus Pleuronema is still not supported.     

Evolution of two distinct phylogenetic lineages of the emerging human pathogen <Emphasis Type="Italic">Mycobacterium ulcerans</Emphasis>

Background  

Comparative genomics has greatly improved our understanding of the evolution of pathogenic mycobacteria such as Mycobacterium tuberculosis. Here we have used data from a genome microarray analysis to explore insertion-deletion (InDel) polymorphism among a diverse strain collection of Mycobacterium ulcerans, the causative agent of the devastating skin disease, Buruli ulcer. Detailed analysis of large sequence polymorphisms in twelve regions of difference (RDs), comprising irreversible genetic markers, enabled us to refine the phylogenetic succession within M. ulcerans, to define features of a hypothetical M. ulcerans most recent common ancestor and to confirm its origin from Mycobacterium marinum.     

Tropical Plant Extracts Modulating the Growth of Mycobacterium ulcerans

Mycobacterium ulcerans, the etiologic agent of Buruli ulcer, has been detected on aquatic plants in endemic tropical regions. Here, we tested the effect of several tropical plant extracts on the growth of M. ulcerans and the closely related Mycobacterium marinum. M. ulcerans and M. marinum were inoculated on Middlebrook 7H11 medium with and without extracts from tropical aquatic plants, including Ammannia gracilis, Crinum calamistratum, Echinodorus africanus, Vallisneria nana and Vallisneria torta. Delay of detection of the first colony and the number of colonies at day 7 (M. marinum) or day 16 (M. ulcerans) were used as endpoints. The first M. ulcerans colonies were detected at 8 ± 0 days on control Middlebrook 7H11 medium, 6.34 ± 0.75 days on A. gracilis-enriched medium (p<0.01), 6 ± 1 days on E. africanus- and V. torta-enriched media (p<0.01), 6 ± 0 days on V. nana-enriched medium (p<0.01) and 5.67 ± 0.47 days on C. calamistratum-enriched medium (p<0.01). Furthermore, the number of detected colonies was significantly increased in C. calamistratum- and E. africanus-enriched media at each time point compared to Middlebrook 7H11 (p<0.05). V. nana- and V. torta-enriched media significantly increased the number of detected colonies starting from day 6 and day 10, respectively (p<0.001). At the opposite, A. gracilis-enriched medium significantly decreased the number of detected colonies starting from day 8 PI (p<0.05). In conclusion, some aquatic plant extracts, could be added as adjuvants to the Middlebrook 7H11 medium for the culturing of M. marinum and M. ulcerans.     

Mycobacterium tuberculosis Promotes Anti-apoptotic Activity of the Macrophage by PtpA Protein-dependent Dephosphorylation of Host GSK3α

Mycobacterium tuberculosis tyrosine phosphatase PtpA inhibits two key cellular events in macrophages required for the elimination of invading organisms, phagosome acidification, and maturation. Kinome analysis revealed multiple PtpA-dependent changes to the phosphorylation status of macrophage proteins upon M. tuberculosis infection. Among those proteins we show that PtpA dephosphorylates GSK3α on amino acid Tyr²⁷⁹, which leads to modulation of GSK3α anti-apoptotic activity, promoting pathogen survival early during infection.     

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes

Klebsiella pneumoniae is an important cause of community‐acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3‐kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella‐containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV‐killed bacteria, the majority of live bacteria did not co‐localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K–Akt–Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down‐regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.     

S-nitrosylation of Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) induces its structural instability

S-nitrosylation is associated with signal transduction and microbicidal activity of nitric oxide (NO). We have recently described the S-nitrosylation of Mycobacterium tuberculosis protein tyrosine phosphatase A, PtpA, an enzyme that plays an important role in mycobacteria survival inside macrophages. This post-translational modification decreases the activity of the enzyme upon modification of a single Cys residue, C53. The aim of the present work was the investigation of the effect of S-nitrosylation in PtpA kinetic parameters, thermal stability and structure. It was observed that the K_M of nitrosylated PtpA was similar to its unmodified form, but the V_max was significantly reduced. In contrast, treatment of PtpA C53A with GSNO, did not alter either K_M or V_max. These results confirmed that PtpA S-nitrosylation occurs specifically in the non-catalytic C53 and that this modification does not affect substrate affinity. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques it was shown that PtpA S-nitrosylation decreased protein thermal stability and promoted a local effect in the surroundings of the C53 residue, which interfered in both protein stability and function.     

Tracking the dynamic interplay between bacterial and host factors during pathogen‐induced vacuole rupture in real time

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen‐induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram‐negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin‐3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high‐content and high‐throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.     

Fragment-based discovery of selective inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

The development of low μM inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported. The most potent of these inhibitors (K_i = 1.4 ± 0.3 μM) was found to be selective when tested against a panel of human tyrosine and dual-specificity phosphatases (11-fold vs the highly homologous HCPtpA, and >70-fold vs all others tested). Modeling the inhibitor-PtpA complexes explained the structure–activity relationships observed in vitro and revealed further possibilities for compound development.     

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.     

Secreted Acid Phosphatase (SapM) of Mycobacterium tuberculosis Is Indispensable for Arresting Phagosomal Maturation and Growth of the Pathogen in Guinea Pig Tissues

Tuberculosis (TB) is responsible for nearly 1.4 million deaths globally every year and continues to remain a serious threat to human health. The problem is further complicated by the growing incidence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), emphasizing the need for the development of new drugs against this disease. Phagosomal maturation arrest is an important strategy employed by Mycobacterium tuberculosis to evade the host immune system. Secretory acid phosphatase (SapM) of M.tuberculosis is known to dephosphorylate phosphotidylinositol 3-phosphate (PI3P) present on phagosomes. However, there have been divergent reports on the involvement of SapM in phagosomal maturation arrest in mycobacteria. This study was aimed at reascertaining the involvement of SapM in phagosomal maturation arrest in M.tuberculosis. Further, for the first time, we have also studied whether SapM is essential for the pathogenesis of M.tuberculosis. By deleting the sapM gene of M.tuberculosis, we demonstrate that MtbΔsapM is defective in the arrest of phagosomal maturation as well as for growth in human THP-1 macrophages. We further show that MtbΔsapM is severely attenuated for growth in the lungs and spleen of guinea pigs and has a significantly reduced ability to cause pathological damage in the host when compared with the parental strain. Also, the guinea pigs infected with MtbΔsapM exhibited a significantly enhanced survival when compared with M.tuberculosis infected animals. The importance of SapM in phagosomal maturation arrest as well as in the pathogenesis of M.tuberculosis establishes it as an attractive target for the development of new therapeutic molecules against tuberculosis.     

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER‐associated compartment termed the Legionella‐containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule‐resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant‐negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P‐positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1‐dependent aggregation of purified, ER‐positive LCVs in vitro. Thus, Sey1/Atl3‐dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.     

Discovery of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B (PtpB) Inhibitors from Natural Products

Protein tyrosine phosphatase B (PtpB) is one of the virulence factors secreted into the host cell by Mycobacterium tuberculosis. PtpB attenuates host immune defenses by interfering with signal transduction pathways in macrophages and, therefore, it is considered a promising target for the development of novel anti-tuberculosis drugs. Here we report the discovery of natural compound inhibitors of PtpB among an in house library of more than 800 natural substances by means of a multidisciplinary approach, mixing in silico screening with enzymatic and kinetics studies and MS assays. Six natural compounds proved to inhibit PtpB at low micromolar concentrations (< 30 µM) with Kuwanol E being the most potent with K _i = 1.6 ± 0.1 µM. To the best of our knowledge, Kuwanol E is the most potent natural compound PtpB inhibitor reported so far, as well as it is the first non-peptidic PtpB inhibitor discovered from natural sources. Compounds herein identified may inspire the design of novel specific PtpB inhibitors.