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Mycobacterium avium subsp. hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread.

ABSTRACT: Mycobacterium avium subsp. hominissuis is an opportunistic intracellular pathogen associated with disease in patients either immunosuppression or chronic lung pathology. Once in the host, M. avium preferentially infects and replicates within the phagocytic cells. The host driven macrophage apoptosis appears to be an essential aspect of innate immunity during bacterial infection; however, the existing evidence suggests that M. avium has evolved adaptive approaches to trigger the phagocyte apoptosis, exit apoptotic cells or via ingestion of infected apoptotic bodies subsequently infect neighboring macrophages. By evaluating 4,000 transposon mutants of M. avium in THP-1 cells, we identified clones that can trigger a new form of early host cell apoptosis, which is only observed upon entry into the "secondary-infected" macrophages. Inactivation of MAVA5_06970 gene lead to significant attenuation in intracellular growth within macrophages and mice, and impaired M. avium to induce rapid apoptosis in the "secondary-infected" cells as measured by Annexin V-FITC detection assay. Complementation of MAVA5_06970 gene corrected the attenuation as well as apoptotic phenotypes. The MAVA5_06970 gene encodes for a secreted protein. Using the pull-down assay and then confirmed with the yeast two-hybrid screen, we found that MAVA5_06970 effector interacts with the Secreted Phosphoprotein 1, the cytokine also known as Osteopontin. This interaction enhances the THP-1 cell apoptosis and, consequently, restricts the production of interleukin-12 that likely may limit the activation of the type I immunity pathway in vivo. This work identified a key virulence effector of M. avium that contributes to the cell-to-cell spread of the pathogen.

SUBMITTER: Danelishvili L

PROVIDER: S-EPMC6177253 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Mycobacterium avium subsp. hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread.

Danelishvili Lia L Rojony Rajoana R Carson Kylee L KL Palmer Amy L AL Rose Sasha J SJ Bermudez Luiz E LE

Virulence 20180101 1

Mycobacterium avium subsp. hominissuis is an opportunistic intracellular pathogen associated with disease in patients either immunosuppression or chronic lung pathology. Once in the host, M. avium preferentially infects and replicates within the phagocytic cells. The host driven macrophage apoptosis appears to be an essential aspect of innate immunity during bacterial infection; however, the existing evidence suggests that M. avium has evolved adaptive approaches to trigger the phagocyte apoptos ...[more]

PMID: 30134761

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Project description:BackgroundMany 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 findingsWe 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/significanceWe 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.

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.

Dataset Information

Mycobacterium avium subsp. hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread.

Publications

Mycobacterium avium subsp. hominissuis effector MAVA5_06970 promotes rapid apoptosis in secondary-infected macrophages during cell-to-cell spread.

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