Temporal genome-wide fitness analysis of Mycobacterium marinum during infection reveals the genetic requirement for virulence and survival in amoebae and microglial cells.

Tuberculosis remains the most pervasive infectious disease and the recent emergence of drug-resistant strains emphasizes the need for more efficient drug treatments. A key feature of pathogenesis, conserved between the human pathogen Mycobacterium tuberculosis and the model pathogen Mycobacterium marinum, is the metabolic switch to lipid catabolism and altered expression of virulence genes at different stages of infection. This study aims to identify genes involved in sustaining viable intracellular infection. We applied transposon sequencing (Tn-Seq) to M. marinum, an unbiased genome-wide strategy combining saturation insertional mutagenesis and high-throughput sequencing. This approach allowed us to identify the localization and relative abundance of insertions in pools of transposon mutants. Gene essentiality and fitness cost of mutations were quantitatively compared between in vitro growth and different stages of infection in two evolutionary distinct phagocytes, the amoeba Dictyostelium discoideum and the murine BV2 microglial cells. In the M. marinum genome, 57% of TA sites were disrupted and 568 genes (10.2%) were essential, which is comparable to previous Tn-Seq studies on M. tuberculosis and M. bovis. Major pathways involved in the survival of M. marinum during infection of D. discoideum are related to DNA damage repair, lipid and vitamin metabolism, the type VII secretion system (T7SS) ESX-1, and the Mce1 lipid transport system. These pathways, except Mce1 and some glycolytic enzymes, were similarly affected in BV2 cells. These differences suggest subtly distinct nutrient availability or requirement in different host cells despite the known predominant use of lipids in both amoeba and microglial cells.IMPORTANCEThe emergence of biochemically and genetically tractable host model organisms for infection studies holds the promise to accelerate the pace of discoveries related to the evolution of innate immunity and the dissection of conserved mechanisms of cell-autonomous defenses. Here, we have used the genetically and biochemically tractable infection model system Dictyostelium discoideum/Mycobacterium marinum to apply a genome-wide transposon-sequencing experimental strategy to reveal comprehensively which mutations confer a fitness advantage or disadvantage during infection and compare these to a similar experiment performed using the murine microglial BV2 cells as host for M. marinum to identify conservation of virulence pathways between hosts.

Assessment of HIV viral load monitoring in remote settings in Vietnam - comparing people who inject drugs to the other patients.

INTRODUCTION: Increasing access to viral load (VL) monitoring is essential to fight HIV epidemics. In remote settings in Vietnam, using dried blood spot (DBS) sampling for specimen collection could improve the situation. Here, people who inject drugs (PWID) represent many newly antiretroviral therapy (ART)-initiated patients. The goals of this evaluation were to evaluate if access to VL monitoring and the rate of virological failure differed between PWID and non-PWID. METHODS: Prospective cohort study of patients newly initiated on ART in remote settings in Vietnam. DBS coverage at 6, 12 and 24 months of ART was investigated. Factors associated with DBS coverage were identified through logistic regression, as were factors associated with virological failure (VL ≥1,000 copies/mL) at 6, 12 and 24 months of ART. RESULTS: Overall 578 patients were enrolled in the cohort, of whom 261 (45%) were PWID. DBS coverage improved from 74.7% to 82.9% between 6 and 24 months of ART (p = 0.001). PWID status was not associated with DBS coverage (p = 0.74), but DBS coverage was lower in patients who were late to clinical visits and in those in WHO stage 4 (p = 0.023 and p = 0.001, respectively). The virological failure rate decreased from 15.8% to 6.6% between 6 and 24 months of ART (p<0.001). In multivariate analysis, PWID were more at risk of failure (p = 0.001), as were patients who were late to clinical visits (p<0.001) and not fully adherent (p<0.001). CONCLUSIONS: Despite training and simple procedures, DBS coverage was not perfect. DBS coverage was not associated with PWID status. Close management is required for effective routine HIV VL monitoring. PWID were more at risk of failure, as were patients who were not fully adherent and patients who were late to clinical visits. Specific interventions targeting these patients are needed to improve their outcomes. Overall, efforts in coordination and communication are essential to improve global HIV care. TRIAL REGISTRATION: Clinical Trial Number: NCT03249493.

Novel Single-Cell and High-Throughput Microscopy Techniques to Monitor Dictyostelium discoideum-Mycobacterium marinum Infection Dynamics.

The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.

Impact of original, B.1.1.7, and B.1.351/P.1 SARS-CoV-2 lineages on vaccine effectiveness of two doses of COVID-19 mRNA vaccines: Results from a nationwide case-control study in France.

BACKGROUND: We aimed to assess the effectiveness of two doses of mRNA COVID-19 vaccines against COVID-19 with the original virus and other lineages circulating in France. METHODS: In this nationwide case-control study, cases were SARS-CoV-2 infected adults with onset of symptoms between 14 February and 3 May 2021. Controls were non-infected adults from a national representative panel matched to cases by age, sex, region, population density and calendar week. Participants completed an online questionnaire on recent activity-related exposures and vaccination history. Information about the infecting virus was based on a screening RT-PCR for either B.1.1.7 or B.1.351/P.1 variants. FINDINGS: Included in our analysis were 7 288 adults infected with the original SARS-CoV-2 virus, 31 313 with the B.1.1.7 lineage, 2 550 with B.1.351/P1 lineages, and 3 644 controls. In multivariable analysis, the vaccine effectiveness (95% confidence interval) seven days after the second dose of mRNA vaccine was estimated at 88% (81-92), 86% (81-90) and 77% (63-86) against COVID-19 with the original virus, the B.1.1.7 lineage, and the B.1.351/P.1 lineages, respectively. Recent (2 to 6 months) history of virologically confirmed SARS-CoV-2 infection was found to be 83% (76-88), 88% (85-91) and 83% (71-90) protective against COVID-19 with the original virus, the B.1.1.7 lineage, and the B.1.351/P.1 lineages, respectively; and more distant (> 6 months) infections were 76% (54-87), 84% (75-90), and 74% (41-89) protective against COVID-19 with the original virus, the B.1.1.7 lineage, and the B.1.351/P.1 lineages, respectively. INTERPRETATION: In real-life settings, two doses of mRNA vaccines proved to be effective against COVID-19 with the original virus, B.1.1.7 lineage and B.1.351/P.1 lineages. FUNDING: Institut Pasteur, Research & Action Emerging Infectious Diseases (REACTing), Fondation de France (Alliance "Tous unis contre le virus").

Zn2+ Intoxication of Mycobacterium marinum during Dictyostelium discoideum Infection Is Counteracted by Induction of the Pathogen Zn2+ Exporter CtpC.

Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum senses toxic levels of Zn2+ and responds by upregulating one of its isoforms of the Zn2+ efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn2+ as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR_1271 is the functional CtpC Zn2+ efflux transporter in M. marinum Infection leads to the accumulation of Zn2+ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn2+ efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn2+ into the MCV, carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn2+ than in wild-type cells, suggesting restriction by other Zn2+-mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn2+IMPORTANCE Microelements are essential for the function of the innate immune system. A deficiency in zinc or copper results in an increased susceptibility to bacterial infections. Zn2+ serves as an important catalytic and structural cofactor for a variety of enzymes including transcription factors and enzymes involved in cell signaling. But Zn2+ is toxic at high concentrations and represents a cell-autonomous immunity strategy that ensures killing of intracellular bacteria in a process called zinc poisoning. The cytosolic and lumenal Zn2+ concentrations result from the balance of import into the cytosol via ZIP influx transporters and efflux via ZnT transporters. Here, we show that Zn2+ poisoning is involved in restricting Mycobacterium marinum infections. Our study extends observations during Mycobacterium tuberculosis infection and explores for the first time how the interplay of ZnT transporters affects mycobacterial infection by impacting Zn2+ homeostasis.

RskA Is a Dual Function Activator-Inhibitor That Controls SigK Activity Across Distinct Bacterial Genera.

It has been previously shown that RskA, the anti-Sigma factor K of Mycobacterium tuberculosis, inhibits SigK and that mutations in RskA promote high expression of the SigK regulon. The latter observation led us to hypothesize that RskA mutations lead to loss of the anti-Sigma factor function. In this report, we used natural and artificial mutations in RskA to determine the basis of the SigK-RskA partnership. Consistent with predictions, the N-terminal cytoplasmic portion of RskA was sufficient on its own to inhibit SigK. Unexpectedly, RskA also served as an activator of SigK. This activation depended on the same N-terminal region and was enhanced by the membrane-extracellular portion of RskA. Based on this, we engineered similar truncations in a Gram-negative bacterium, namely Yersinia enterocolitica. Again, we observed that, with specific alterations of RskA, we were able to enhance SigK activity. Together these results support an alternative mechanism of anti-Sigma factor function, that we could term modulator (activator-inhibitor) in both Actinobacteria and Gram-negative bacteria, suggesting that Sigma factor activation by anti-Sigma factors could be under-recognized.

Feature of Adhesins Produced by Human Clinical Isolates of Mycobacterium intracellulare, Mycobacterium intracellulare subsp. chimaera and Closely Related Species.

The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare. They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.

Localization of all four ZnT zinc transporters in Dictyostelium and impact of ZntA and ZntB knockout on bacteria killing.

Professional phagocytes have developed an extensive repertoire of autonomous immunity strategies to ensure killing of bacteria. Besides phagosome acidification and the generation of reactive oxygen species, deprivation of nutrients and the lumenal accumulation of toxic metals are essential to kill ingested bacteria or inhibit the growth of intracellular pathogens. Here, we used the soil amoeba Dictyostelium discoideum, a professional phagocyte that digests bacteria for nutritional purposes, to decipher the role of zinc poisoning during phagocytosis of nonpathogenic bacteria and visualize the temporal and spatial dynamics of compartmentalized, free zinc using fluorescent probes. Immediately after particle uptake, zinc is delivered to phagosomes by fusion with 'zincosomes' of endosomal origin, and also by the action of one or more zinc transporters. We localized the four Dictyostelium ZnT transporters to endosomes, the contractile vacuole and the Golgi complex, and studied the impact of znt knockouts on zinc homeostasis. We show that zinc is delivered into the lumen of Mycobacterium smegmatis-containing vacuoles, and that Escherichia coli deficient in the zinc efflux P1B-type ATPase ZntA are killed faster than wild-type bacteria.

When Dicty Met Myco, a (Not So) Romantic Story about One Amoeba and Its Intracellular Pathogen.

In recent years, Dictyostelium discoideum has become an important model organism to study the cell biology of professional phagocytes. This amoeba not only shares many molecular features with mammalian macrophages, but most of its fundamental signal transduction pathways are conserved in humans. The broad range of existing genetic and biochemical tools, together with its suitability for cell culture and live microscopy, make D. discoideum an ideal and versatile laboratory organism. In this review, we focus on the use of D. discoideum as a phagocyte model for the study of mycobacterial infections, in particular Mycobacterium marinum. We look in detail at the intracellular cycle of M. marinum, from its uptake by D. discoideum to its active or passive egress into the extracellular medium. In addition, we describe the molecular mechanisms that both the mycobacterial invader and the amoeboid host have developed to fight against each other, and compare and contrast with those developed by mammalian phagocytes. Finally, we introduce the methods and specific tools that have been used so far to monitor the D. discoideum-M. marinum interaction.

Eat Prey, Live: Dictyostelium discoideum As a Model for Cell-Autonomous Defenses.

The soil-dwelling social amoeba Dictyostelium discoideum feeds on bacteria. Each meal is a potential infection because some bacteria have evolved mechanisms to resist predation. To survive such a hostile environment, D. discoideum has in turn evolved efficient antimicrobial responses that are intertwined with phagocytosis and autophagy, its nutrient acquisition pathways. The core machinery and antimicrobial functions of these pathways are conserved in the mononuclear phagocytes of mammals, which mediate the initial, innate-immune response to infection. In this review, we discuss the advantages and relevance of D. discoideum as a model phagocyte to study cell-autonomous defenses. We cover the antimicrobial functions of phagocytosis and autophagy and describe the processes that create a microbicidal phagosome: acidification and delivery of lytic enzymes, generation of reactive oxygen species, and the regulation of Zn2+, Cu2+, and Fe2+ availability. High concentrations of metals poison microbes while metal sequestration inhibits their metabolic activity. We also describe microbial interference with these defenses and highlight observations made first in D. discoideum. Finally, we discuss galectins, TNF receptor-associated factors, tripartite motif-containing proteins, and signal transducers and activators of transcription, microbial restriction factors initially characterized in mammalian phagocytes that have either homologs or functional analogs in D. discoideum.

Novel feature of Mycobacterium avium subsp. paratuberculosis, highlighted by characterization of the heparin-binding hemagglutinin adhesin.

Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.

Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. FINDINGS: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. CONCLUSIONS: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays.

Inter- and intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne's disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as 'Sheep' or 'S-type' and 'Cattle' or 'C-type'. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis. RESULTS: The presence of LSP(A)4 and absence of LSP(A)20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III. CONCLUSION: This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains.

Characterization of the Mycobacterium avium subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which reacts with sera from patients with Crohn's disease.

Mycobacterium avium subsp. paratuberculosis (Map) causes a chronic enteric disease in ruminants, called paratuberculosis or Johne's disease. The current model proposes that after ingestion by the host, Map crosses the intestinal barrier via internalization by the M cells. Experimental observations suggest, however, that Map may also transcytose the intestinal wall via the enterocytes, but the mechanisms involved in this process remain poorly understood. Cytoadherence assays performed on epithelial cells with Map revealed that the addition of laminin to the cell culture increases adhesion. A Map protein was isolated by heparin-Sepharose chromatography and identified as a laminin-binding protein like. The gene encoding this protein named Lbp/Hlp was identified in the Map genome sequence at locus MAP3024 (annotated Hup B). The deduced Map Lbp/Hlp amino acid sequence reveals 80% identity with that reported for other mycobacteria. The C-terminal domain involved in adhesion is mainly composed of arginine and lysine residues modified by methylation. In vitro tests demonstrated that recombinant Lbp/Hlp binds laminin, heparin, collagen and epithelial cells. Interestingly, we found that this adhesin corresponds to the antigen described as the target of pANCA and serum antibodies of patients with Crohn's disease.