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MAV_4644 Interaction with the Host Cathepsin Z Protects Mycobacterium avium subsp. hominissuis from Rapid Macrophage Killing.

ABSTRACT: Mycobacterium avium subspecies hominissuis (MAH) is an opportunistic pathogen that is ubiquitous in the environment and often isolated from faucets and showerheads. MAH mostly infects humans with an underlying disease, such as chronic pulmonary disorder, cystic fibrosis, or individuals that are immunocompromised. In recent years, MAH infections in patients without concurrent disease are increasing in prevalence as well. This pathogen is resistant to many antibiotics due to the impermeability of its envelope and due to the phenotypic resistance established within the host macrophages, making difficult to treat MAH infections. By screening a MAH transposon library for mutants that are susceptible to killing by reactive nitrogen intermediaries, we identified the MAV_4644 (MAV_4644:Tn) gene knockout clone that was also significantly attenuated in growth within the host macrophages. Complementation of the mutant restored the wild-type phenotype. The MAV_4644 gene encodes a dual-function protein with a putative pore-forming function and ADP-ribosyltransferase activity. Protein binding assay suggests that MAV_4644 interacts with the host lysosomal peptidase cathepsin Z (CTSZ), a key regulator of the cell signaling and inflammation. Pathogenic mycobacteria have been shown to suppress the action of many cathepsins to establish their intracellular niche. Our results demonstrate that knocking-down the cathepsin Z in human macrophages rescues the attenuated phenotype of MAV_4644:Tn clone. Although, the purified cathepsin Z by itself does not have any killing effect on MAH, it contributes to bacterial killing in the presence of the nitric oxide (NO). Our data suggest that the cathepsin Z is involved in early macrophage killing of MAH, and the virulence factor MAV_4644 protects the pathogen from this process.

SUBMITTER: Lewis MS

PROVIDER: S-EPMC6560410 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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MAV_4644 Interaction with the Host Cathepsin Z Protects Mycobacterium avium subsp. hominissuis from Rapid Macrophage Killing.

Lewis Matthew S MS Danelishvili Lia L Rose Sasha J SJ Bermudez Luiz E LE

Microorganisms 20190521 5

Mycobacterium avium subspecies hominissuis (MAH) is an opportunistic pathogen that is ubiquitous in the environment and often isolated from faucets and showerheads. MAH mostly infects humans with an underlying disease, such as chronic pulmonary disorder, cystic fibrosis, or individuals that are immunocompromised. In recent years, MAH infections in patients without concurrent disease are increasing in prevalence as well. This pathogen is resistant to many antibiotics due to the impe ...[more]

PMID: 31117286

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Project description:Nontuberculous mycobacterial infections caused by the opportunistic pathogen Mycobacterium avium subsp. hominissuis (MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence and in vitro growth and organized ?3,500 identified transposon mutants for hypothesis-driven research. The majority (96%) of the genes we identified as essential for MAH in vitro had a mutual ortholog in the related and highly virulent Mycobacterium tuberculosis (Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. More than 97% of the MAH virulence genes had a mutual ortholog in Mtb Finally, we validated novel genes required for successful MAH infection: one encoding a probable major facilitator superfamily (MFS) transporter and another encoding a hypothetical protein located in the immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection.IMPORTANCE Pulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the Mycobacterium avium complex (MAC), whereof >90% are due to Mycobacterium avium subsp. hominissuis (MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12?months. To control MAH by improved therapy, prevention, and diagnostics, we need to understand the underlying mechanisms of infection. Here, we provide crucial insights into MAH's global genetic requirements for growth and infection. We find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen M. tuberculosis (Mtb). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment or translate to broader treatment of related mycobacterial diseases.

Project description:BACKGROUND:Many efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections. METHODOLOGY/PRINCIPAL FINDINGS:We performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR). The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs. CONCLUSIONS/SIGNIFICANCE:We show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response. In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively.

Dataset Information

MAV_4644 Interaction with the Host Cathepsin Z Protects Mycobacterium avium subsp. hominissuis from Rapid Macrophage Killing.

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MAV_4644 Interaction with the Host Cathepsin Z Protects <i>Mycobacterium avium</i> subsp. <i>hominissuis</i> from Rapid Macrophage Killing.

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