Primary cutaneous infections with non-tuberculous mycobacteria: a report of 6 cases.

BACKGROUND: The incidence of non-tuberculous mycobacterium infection has shown a gradual increasing trend in recent years, among which cutaneous manifestations as an important aspect. This study aimed to describe the clinical features and microbiological findings in 6 cases of primary cutaneous nontuberculous mycobacterium infection. METHODS: In this retrospective study from June 2021 to June 2022, the clinical data and microbiological results of six cases diagnosed with primary cutaneous non-tuberculous mycobacterium infection in department of dermatology, Hangzhou Third People's Hospital were analyzed. RESULTS: All six cases were primary cutaneous non-tuberculous mycobacterium infections, four of which had a history of trauma or exposure, and two had an underlying disease that could lead to compromised immunity. All patients presented with erythema nodular skin lesions, four on the upper or lower extremities, one on the face, and one on the right hip. The histopathological findings of five patients who underwent biopsy were granulomatous inflammatory changes with mixed infiltration. Laboratory cultures using tissue or tissue fluid were all successful, including four Mycobacterium marinum, one Mycobacterium abscessus, and one Mycobacterium avium. Metagenomics next-generation sequencing detected results consistent with culture colonies in only two cases. With the exception of case 4, all patients responded well to oral medication, with a course of treatment ranging from 4 months to 1 year, and the prognosis was good. CONCLUSIONS: The clinical features of primary cutaneous non-tuberculous mycobacterium infection are often lacking in specificity, and the identification of related strains is difficult for a variety of reasons. Although the results of metagenomics next-generation sequencing are useful for pathogen spectrum identification, its diagnostic value should be carefully reevaluated under certain circumstances. Patients with suspected triggers who do not respond well to conventional treatments should be suspected as atypical infection and potential immunosuppression. If diagnosed and treated promptly, the prognosis of primary cutaneous non-tuberculous mycobacterium infection is generally good.

PE/PPE Proteome and ESX-5 Substrate Spectrum in Mycobacterium marinum.

PE/PPE proteins secreted by the ESX-5 type VII secretion system constitute a major protein repertoire in pathogenic mycobacteria and are essential for bacterial survival, pathogenicity, and host-pathogen interaction; however, little is known about their expression and secretion. The scarcity of arginine and lysine residues in PE/PPE protein sequences and the high homology of their N-terminal domains limit protein identification using classical trypsin-based proteomic methods. This study used endoproteinase AspN and trypsin to characterize the proteome of Mycobacterium marinum. Twenty-seven PE/PPE proteins were uniquely identified in AspN digests, especially PE_PGRS proteins. These treatments allowed the identification of approximately 50% of the PE/PPE pool encoded in the genome. Moreover, EspG5 pulldown assays retrieved 44 ESX-5-associated PPE proteins, covering 85% of the PPE pool in the identified proteome. The identification of PE/PE_PGRS proteins in the EspG5 interactome suggested the presence of PE-PPE pairs. The correlation analysis between protein abundance and phylogenetic relationships found potential PE/PPE pairs, indicating the presence of multiple PE/PE_PGRS partners in one PPE. We validated that EspG5 interacted with PPE31 and PPE32 and mapped critical residues for complex formation. The modified proteomic platform increases the coverage of PE/PPE proteins and elucidates the expression and localization of these proteins.

Mycobacterium marinum MMAR_0267-regulated copper utilization facilitates bacterial escape from phagolysosome.

The host limits Mycobacterium tuberculosis (Mtb) by enriching copper in high concentrations. This research investigates how Mtb escapes copper stress. The membrane protein encoded by Mtb Rv0102, when its homolog in M. smegmatis (MSMEG_4702) was knocked out, resulted in a fourfold decrease in intracellular copper levels and enhanced tolerance to elevated extracellular copper concentrations. Similarly, knockout mutants of its homolog in M. marinum (MMAR_0267) showed increased virulence in zebrafish and higher bacterial load within macrophages. In THP-1 cells infected with MMAR_0267 deletion mutants, the intracellular survival of these mutants increased, along with reduced THP-1 cell apoptosis. Deficiency in copper down-regulated the transcriptional level of the virulence factor CFP-10 in M. marinum, suppressed cytosolic signaling via the macrophage STING pathway, leading to decreased production of IFN-ß and reduced cell apoptosis. In conclusion, these findings highlight the significant impact of copper on the survival and reproduction of mycobacteria, underscoring the importance of studying mycobacterial adaptation mechanisms in copper-rich environments.

Cytosolic serpins act in a cytoprotective feedback loop that limits ESX-1-dependent death of Mycobacterium marinum-infected macrophages.

Serine protease inhibitors (serpins) constitute the largest family of protease inhibitors expressed in humans, but their role in infection remains largely unexplored. In infected macrophages, the mycobacterial ESX-1 type VII secretion system permeabilizes internal host membranes and causes leakage into the cytosol of host DNA, which induces type I interferon (IFN) production via the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) surveillance pathway, and promotes infection in vivo. Using the Mycobacterium marinum infection model, we show that ESX-1-mediated type I IFN signaling in macrophages selectively induces the expression of serpina3f and serpina3g, two cytosolic serpins of the clade A3. The membranolytic activity of ESX-1 also caused leakage of cathepsin B into the cytosol where it promoted cell death, suggesting that the induction of type I IFN comes at the cost of lysosomal rupture and toxicity. However, the production of cytosolic serpins suppressed the protease activity of cathepsin B in this compartment and thus limited cell death, a function that was associated with increased bacterial growth in infected mice. These results suggest that cytosolic serpins act in a type I IFN-dependent cytoprotective feedback loop to counteract the inevitable toxic effect of ESX-1-mediated host membrane rupture. IMPORTANCE: The ESX-1 type VII secretion system is a key virulence determinant of pathogenic mycobacteria. The ability to permeabilize host cell membranes is critical for several ESX-1-dependent virulence traits, including phagosomal escape and induction of the type I interferon (IFN) response. We find that it comes at the cost of lysosomal leakage and subsequent host cell death. However, our results suggest that ESX-1-mediated type I IFN signaling selectively upregulates serpina3f and serpina3g and that these cytosolic serpins limit cell death caused by cathepsin B that has leaked into the cytosol, a function that is associated with increased bacterial growth in vivo. The ability to rupture host membranes is widespread among bacterial pathogens, and it will be of interest to evaluate the role of cytosolic serpins and this type I IFN-dependent cytoprotective feedback loop in the context of human infection.

Diagnosis of Mycobacterium marinum Infection by Metagenomic next-generation sequencing.

BACKGROUND: In December 2023, our hospital confirmed a case of finger infection with Mycobacterium marinum. The patient sought medical attention at our hospital due to a hard scratch on her left middle finger, which was red, swollen, and ulcerated for one month. PHYSICAL EXAMINATION: A lesion of approximately 1.5 cm x 2 cm in the patient's left middle finger, surrounded by redness and swelling, unclear boundaries, surface rupture, partial scabbing, and no tenderness during compression. She was treated at the previous clinic, common infectious diseases were considered, and was given intravenous infusion treatment: cefotaxime and clarithromycin, and erythromycin ointment was applied externally. She came to our hospital after poor treatment results. The patient has had hypertension for 3 years, no other systemic diseases, no similar medical history among family members, no history of drug or food allergies. METHODS: Clean the wound and remove the scab from the affected area, and use a surgical blade to scrape off necrotic tissue. Send the scraped tissue for pathogen testing: tissue bacterial culture+identification (matrix assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF), tissue acid fast staining, and tissue metagenomic next-generation sequencing (mNGS). Other auxiliary examinations: blood routine, urine routine, blood fat, liver function, and kidney function. RESULTS: Tissue bacterial culture+identification: growth of Mycobacterium marinum; Acid fast staining of tissue: positive; Tissue mNGS: Mycobacterium marinum. Clinical treatment plan: clarithromycin 0.5 g bid po+rifampicin 0.45 g qd po+5-aminolevulinic acid photodynamic therapy (ALA-PDT) qw+boric acid wash wet compress tid. After 14 days of treatment, the area of redness and swelling significantly decreased, and the degree of redness and swelling was significantly reduced compared to admission. The degree of ulcer edge protrusion was also reduced compared to admission. There was a small amount of exudation from the wound, and no necrotic tissue was observed. The patient improved and was discharged. CONCLUSIONS: This article reports a case of finger infection with Mycobacterium marinum. Mycobacterium marinum was quickly and accurately identified by mNGS, and reasonable treatment measures were adopted clinically. The patient improved and was discharged. This study has important reference significance for the clinical diagnosis and treatment of Mycobacterium infection. In addition, mNGS as a novel detection method has considerable prospects for rapid diagnosis of pathogens.

The regulatory functions of ESX-1 substrates, EspE and EspF, are separable from secretion.

Pathogenic mycobacteria are a significant global health burden. The ESX-1 secretion system is essential for mycobacterial pathogenesis. The secretion of ESX-1 substrates is required for phagosomal lysis, which allows the bacteria to enter the macrophage cytoplasm, induce a Type I IFN response, and spread to new host cells. EspE and EspF are dual-functioning ESX-1 substrates. Inside the mycobacterial cell, they regulate transcription of ESX-1-associated genes. Following secretion, EspE and EspF are essential for lytic activity. The link between EspE/F secretion and regulatory function has not been investigated. We investigated the relationship between EspE and EspF using molecular genetics in Mycobacterium marinum, a non-tuberculous mycobacterial species that serves as an established model for ESX-1 secretion and function in Mycobacterium tuberculosis. Our data support that EspE and EspF, which require each other for secretion, directly interact. The disruption of the predicted protein-protein interaction abrogates hemolytic activity and secretion but does not impact their gene regulatory activities in the mycobacterial cell. In addition, we predict a direct protein-protein interaction between the EsxA/EsxB heterodimer and EspF. Our data support that the EspF/EsxA interaction is also required for hemolytic activity and EspE secretion. Our study sheds light on the intricate molecular mechanisms governing the interactions between ESX-1 substrates, regulatory function, and ESX-1 secretion, moving the field forward.IMPORTANCETuberculosis (TB), caused by Mycobacterium tuberculosis, is a historical and pervasive disease responsible for millions of deaths annually. The rise of antibiotic and treatment-resistant TB, as well as the rise of infection by non-tuberculous mycobacterial species, calls for a better understanding of pathogenic mycobacteria. The ESX-1 secreted substrates, EspE and EspF, are required for mycobacterial virulence and may be responsible for phagosomal lysis. This study focuses on the mechanism of EspE and EspF secretion from the mycobacterial cell.

Successful diagnosis of Mycobacterium marinum infection by mNGS in a patient with Human Immunodeficiency Virus: a case report.

INTRODUCTION: Mycobacterium marinum infection rarely occurs and has atypical symptoms. It is challenging to distinguish disseminated M. marinum infection from multifocal dermatosis caused by other factors clinically. CASE PRESENTATION: Herein, we reported a 68-year-old male patient with Human Immunodeficiency Virus (HIV) who presented redness and swelling in his left hand after being stabbed by marine fish for over 2 months. Mycobacterium tuberculosis infection was considered according to biochemical and pathological examinations, while empirical anti-infection treatment was ineffective. RESULTS: The metagenomic next-generation sequencing (mNGS) detected a large amount of M. marinum sequences, and the patient was finally diagnosed with M. marinum infection. After one month of combination therapy with ethambutol, rifabutin, moxifloxacin, and linezolid, the swelling disappeared significantly. In this case, the successful application of mNGS in diagnosing and treating M. marinum infection has improved the understanding of the microbe both in the laboratory and clinically, especially in patients with HIV. CONCLUSIONS: For diseases with atypical symptoms or difficulty in determining the pathogens, mNGS is suggested in clinical procedures for rapid and accurate diagnosis and treatment.

Vitamin B12 uptake across the mycobacterial outer membrane is influenced by membrane permeability in Mycobacterium marinum.

Vitamin B12 (B12) serves as a critical cofactor within mycobacterial metabolism. While some pathogenic strains can synthesize B12 de novo, others rely on host-acquired B12. In this investigation, we studied the transport of vitamin B12 in Mycobacterium marinum using B12-auxotrophic and B12-sensitive strains by deleting metH or metE, respectively. These two enzymes rely on B12 in different ways to function as methionine synthases. We used these strains to select mutants affecting B12 scavenging and confirmed their phenotypes during growth experiments in vitro. Our analysis of B12 uptake mechanisms revealed that membrane lipids and cell wall integrity play an essential role in cell envelope transport. Furthermore, we identified a potential transcription regulator that responds to B12. Our study demonstrates that M. marinum can take up exogenous B12 and that altering mycobacterial membrane integrity affects B12 uptake. Finally, during zebrafish infection using B12-auxotrophic and B12-sensitive strains, we found that B12 is available for virulent mycobacteria in vivo.IMPORTANCEOur study investigates how mycobacteria acquire essential vitamin B12. These microbes, including those causing tuberculosis, face challenges in nutrient uptake due to their strong outer layer. We focused on Mycobacterium marinum, similar to TB bacteria, to uncover its vitamin B12 absorption. We used modified strains unable to produce their own B12 and discovered that M. marinum can indeed absorb it from the environment, even during infections. Changes in the outer layer composition affect this process, and genes related to membrane integrity play key roles. These findings illuminate the interaction between mycobacteria and their environment, offering insights into combatting diseases like tuberculosis through innovative strategies. Our concise research underscores the pivotal role of vitamin B12 in microbial survival and its potential applications in disease control.

The loss of the PDIM/PGL virulence lipids causes differential secretion of ESX-1 substrates in Mycobacterium marinum.

The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis. Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection. The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure phthiocerol dimycocerosate (PDIM)- and phenolic glycolipid (PGL)-dependent protein secretion in M. marinum, a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals. Importantly, M. marinum is a well-established model for mycobacterial pathogenesis. Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups. Loss of PDIM differentially impacted secretion of Group III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggest that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains.IMPORTANCEMycobacterium tuberculosis, the cause of human tuberculosis, killed an estimated 1.3 million people in 2022. Non-tubercular mycobacterial species cause acute and chronic human infections. Understanding how these bacteria cause disease is critical. Lipids in the cell envelope are essential for mycobacteria to interact with the host and promote disease. Strains lacking outer lipids are attenuated for infection, but the reasons are unclear. Our research aims to identify a mechanism for attenuation of mycobacterial strains without the PDIM and PGL outer lipids in M. marinum. These findings will enhance our understanding of the importance of lipids in pathogenesis and how these lipids contribute to other established virulence mechanisms.

Uncovering the roles of Mycobacterium tuberculosis melH in redox and bioenergetic homeostasis: implications for antitubercular therapy.

Mycobacterium tuberculosis (Mtb), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The melH gene in Mtb and Mycobacterium marinum (Mm) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that melH encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of melH in Mm resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of whiB3, a universal stress sensor. The absence of melH also resulted in reduced intracellular levels of NAD+, NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon source dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of melH in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria. IMPORTANCE: This study unveils the pivotal role played by the melH gene in Mycobacterium tuberculosis and in Mycobacterium marinum in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, para-hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of melH. Moreover, changes in crucial cofactors responsible for electron transfer highlighted melH's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of melH as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.

Structural determination and characterisation of the CYP105Q4 cytochrome P450 enzyme from Mycobacterium marinum.

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.

Mycobacterium tuberculosis SecA2-dependent activation of host Rig-I/MAVs signaling is not conserved in Mycobacterium marinum.

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-ß secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a ΔsecA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the ΔsecA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the ΔsecA2 strain. Therefore, to better examine secreted IFN-ß levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-ß to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-ß from macrophages infected with the ΔsecA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-ß response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.

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.

Recruitment of both the ESCRT and autophagic machineries to ejecting Mycobacterium marinum.

Cytosolic Mycobacterium marinum are ejected from host cells such as macrophages or the amoeba Dictyostelium discoideum in a non-lytic fashion. As described previously, the autophagic machinery is recruited to ejecting bacteria and supports host cell integrity during egress. Here, we show that the ESCRT machinery is also recruited to ejecting bacteria, partially dependent on an intact autophagic pathway. As such, the AAA-ATPase Vps4 shows a distinct localization at the ejectosome structure in comparison to fluorescently tagged Vps32, Tsg101 and Alix. Along the bacterium engaged in ejection, ESCRT and the autophagic component Atg8 show partial colocalization. We hypothesize that both, the ESCRT and autophagic machinery localize to the bacterium as part of a membrane damage response, as well as part of a "frustrated autophagosome" that is unable to engulf the ejecting bacterium.

Rapid diagnosis of Mycobacterium marinum infection using targeted nanopore sequencing: a case report.

Mycobacterium marinum (M. marinum) is a non-tuberculous mycobacterium (NTM) that can cause infectious diseases in aquatic animals and humans. Culture-based pathogen detection is the gold standard for diagnosing NTM infection. However, this method is time-consuming and has low positivity rates for fastidious organisms. Oxford Nanopore MinION sequencing is an emerging third-generation sequencing technology that can sequence DNA or RNA directly in a culture-independent manner and offers rapid microbial identification. Further benefits include low cost, short turnaround time, long read lengths, and small equipment size. Nanopore sequencing plays a crucial role in assessing drug resistance, clinical identification of microbes, and monitoring infectious diseases. Some reports on Mycobacterium tuberculosis (MTB) using nanopore sequencing have been published, however, there are few reports on NTM, such as M. marinum. Here, we report the use of nanopore sequencing for the diagnosis of M. marinum.

Mycobacterium tuberculosis ß-lactamase variant reduces sensitivity to ampicillin/avibactam in a zebrafish-Mycobacterium marinum model of tuberculosis.

The ß-lactamase of Mycobacterium tuberculosis, BlaC, hydrolyzes ß-lactam antibiotics, hindering the use of these antibiotics for the treatment of tuberculosis. Inhibitors, such as avibactam, can reversibly inhibit the enzyme, allowing for the development of combination therapies using both antibiotic and inhibitor. However, laboratory evolution studies using Escherichia coli resulted in the discovery of single amino acid variants of BlaC that reduce the sensitivity for inhibitors or show higher catalytic efficiency against antibiotics. Here, we tested these BlaC variants under more physiological conditions using the M. marinum infection model of zebrafish, which recapitulates hallmark features of tuberculosis, including the intracellular persistence of mycobacteria in macrophages and the induction of granuloma formation. To this end, the M. tuberculosis blaC gene was integrated into the chromosome of a blaC frameshift mutant of M. marinum. Subsequently, the resulting strains were used to infect zebrafish embryos in order to test the combinatorial effect of ampicillin and avibactam. The results show that embryos infected with an M. marinum strain producing BlaC show lower infection levels after treatment than untreated embryos. Additionally, BlaC K234R showed higher infection levels after treatment than those infected with bacteria producing the wild-type enzyme, demonstrating that the zebrafish host is less sensitive to the combinatorial therapy of ß-lactam antibiotic and inhibitor. These findings are of interest for future development of combination therapies to treat tuberculosis.

Identification of Mycobacterium pseudoshottsii in the Eastern Mediterranean.

Among the numerous pathogenic nontuberculous mycobacteria (NTM), which may cause disease in both poikilothermic and homoeothermic organisms, members of the unique clade Mycobacterium ulcerans/Mycobacterium marinum (MuMC) may cause disease in both fish and humans. Here, we describe the emergence of Mycobacterium pseudoshottsii, one of the four MuMC members, in Israel. For many years, M. marinum was the dominant NTM that was diagnosed in Israel as a fish pathogen. To the best of our knowledge, this is the first isolation and genomic characterization of M. pseudoshottsii infecting edible fish from two different fish species farmed in offshore sea cages in the eastern Mediterranean as well as in a recirculating aquaculture system in Israel. We compared the M. pseudoshottsii whole-genome sequences to all available genomic sequences of MuMC in free, publicly accessible databases. IMPORTANCE Mycobacterium pseudoshottsii was first detected in 1997 in the USA, infecting wild striped bass (Morone saxatilis). Since then, several reports from different countries worldwide have shown its capacity to become established in new regions as well as its pathogenicity to saltwater and euryhaline finfish of different genera. Our phylogenetic analysis revealed that the Mycobacterium ulcerans/Mycobacterium marinum clade (MuMC) is divided into two main branches: one that includes M. marinum and M. pseudoshottsii, and the second, which includes other M. marinum isolates as well as two isolates of M. shottsii. Our results reinforce the proposition that the geographical distribution of M. pseudoshottsii is much more extensive than is commonly believed. The emergence of M. pseudoshottsii in different parts of the world and its pathogenic traits that affect finfish of different genera may be a cause for concern among fish farmers, researchers, and environmental organizations.

Induction of IL-32 in the immune response of keratinocytes to Mycobacterium marinum infection.

Keratinocytes are the predominant cell type in the skin epidermis, and they not only protect the skin from the influence of external physical factors but also function as an immune barrier against microbial invasion. However, little is known regarding the immune defence mechanisms of keratinocytes against mycobacteria. Here, we performed single-cell RNA sequencing (scRNA-seq) on skin biopsy samples from patients with Mycobacterium marinum infection and bulk RNA sequencing (bRNA-seq) on M. marinum-infected keratinocytes in vitro. The combined analysis of scRNA-seq and bRNA-seq data revealed that several genes were upregulated in M. marinum-infected keratinocytes. Further in vitro validation of these genes by quantitative polymerase chain reaction and western blotting assay confirmed the induction of IL-32 in the immune response of keratinocytes to M. marinum infection. Immunohistochemistry also showed the high expression of IL-32 in patients' lesions. These findings suggest that IL-32 induction is a possible mechanism through which keratinocytes defend against M. marinum infection; this could provide new targets for the immunotherapy of chronic cutaneous mycobacterial infections.

Intragranuloma Accumulation and Inflammatory Differentiation of Neutrophils Underlie Mycobacterial ESX-1-Dependent Immunopathology.

The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum. ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M. marinum infection model, we identify neutrophils and Ly6C+MHCII+ monocytes as the main cellular reservoirs for the bacteria. We show that ESX-1 promotes intragranuloma accumulation of neutrophils and that neutrophils have a previously unrecognized required role in executing ESX-1-mediated pathology. To explore if ESX-1 also regulates the function of recruited neutrophils, we performed a single-cell RNA-sequencing analysis that indicated that ESX-1 drives newly recruited uninfected neutrophils into an inflammatory phenotype via an extrinsic mechanism. In contrast, monocytes restricted the accumulation of neutrophils and immunopathology, demonstrating a major host-protective function for monocytes specifically by suppressing ESX-1-dependent neutrophilic inflammation. Inducible nitric oxide synthase (iNOS) activity was required for the suppressive mechanism, and we identified Ly6C+MHCII+ monocytes as the main iNOS-expressing cell type in the infected tissue. These results suggest that ESX-1 mediates immunopathology by promoting neutrophil accumulation and phenotypic differentiation in the infected tissue, and they demonstrate an antagonistic interplay between monocytes and neutrophils by which monocytes suppress host-detrimental neutrophilic inflammation. IMPORTANCE The ESX-1 type VII secretion system is required for virulence of pathogenic mycobacteria, including Mycobacterium tuberculosis. ESX-1 interacts with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. We demonstrate that ESX-1 promotes immunopathology by driving intragranuloma accumulation of neutrophils, which upon arrival adopt an inflammatory phenotype in an ESX-1-dependent manner. In contrast, monocytes limited the accumulation of neutrophils and neutrophil-mediated pathology via an iNOS-dependent mechanism, suggesting a major host-protective function for monocytes specifically by restricting ESX-1-dependent neutrophilic inflammation. These findings provide insight into how ESX-1 promotes disease, and they reveal an antagonistic functional relationship between monocytes and neutrophils that might regulate immunopathology not only in mycobacterial infection but also in other infections as well as in inflammatory conditions and cancer.