mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity.

Necrosis of macrophages in the granuloma, the hallmark immunological structure of tuberculosis, is a major pathogenic event that increases host susceptibility. Through a zebrafish forward genetic screen, we identified the mTOR kinase, a master regulator of metabolism, as an early host resistance factor in tuberculosis. We found that mTOR complex 1 protects macrophages from mycobacterium-induced death by enabling infection-induced increases in mitochondrial energy metabolism fueled by glycolysis. These metabolic adaptations are required to prevent mitochondrial damage and death caused by the secreted mycobacterial virulence determinant ESAT-6. Thus, the host can effectively counter this early critical mycobacterial virulence mechanism simply by regulating energy metabolism, thereby allowing pathogen-specific immune mechanisms time to develop. Our findings may explain why Mycobacterium tuberculosis, albeit humanity's most lethal pathogen, is successful in only a minority of infected individuals.

An ancestral mycobacterial effector promotes dissemination of infection.

The human pathogen Mycobacterium tuberculosis typically causes lung disease but can also disseminate to other tissues. We identified a M. tuberculosis (Mtb) outbreak presenting with unusually high rates of extrapulmonary dissemination and bone disease. We found that the causal strain carried an ancestral full-length version of the type VII-secreted effector EsxM rather than the truncated version present in other modern Mtb lineages. The ancestral EsxM variant exacerbated dissemination through enhancement of macrophage motility, increased egress of macrophages from established granulomas, and alterations in macrophage actin dynamics. Reconstitution of the ancestral version of EsxM in an attenuated modern strain of Mtb altered the migratory mode of infected macrophages, enhancing their motility. In a zebrafish model, full-length EsxM promoted bone disease. The presence of a derived nonsense variant in EsxM throughout the major Mtb lineages 2, 3, and 4 is consistent with a role for EsxM in regulating the extent of dissemination.

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.

Resolving exit strategies of mycobacteria in Dictyostelium discoideum by combining high-pressure freezing with 3D-correlative light and electron microscopy.

The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium-containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well-established light and advanced electron microscopy techniques in this system, a correlative approach integrating both methods with near-native ultrastructural preservation is currently lacking. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D-correlative light and electron microscopy. This approach includes high-pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens.

Cutaneous infection due to Mycobacterium marinum: A series of four cases from Kerala, India.

Mycobacterium marinum is a non-tuberculous mycobacterium which can be found in naturally occurring, non-chlorinated water sources and is a known pathogen that affects fish. In humans, M. marinum typically results in cutaneous lesions, it can occasionally lead to more invasive disorders. We discuss four cases of M. marinum-related cutaneous infections examined in a tertiary care facility. We want to draw attention to the challenges of accurately diagnosing this infection, stress the significance of having a high level of clinical suspicion in order to identify it, and discuss the available treatment choices.

Proposal of Paenimyroides marinum (Song et al. 2013) comb. nov. to replace the illegitimate name Paenimyroides aquimaris (García-López et al. 2020) Zhang et al. 2023.

In this article, the author addresses the issue of nomenclatural illegitimacy of Paenimyroides aquimaris (García-López et al. 2020) Zhang et al. 2023. This name was formed without re-establishment of the earlier legitimate epithet marinum and should be considered to be illegitimate according to Rule 41a. As required by Rule 54, the author proposes Paenimyroides marinum (Song et al. 2013) as a new combination to replace the illegitimate name Paenimyroides aquimaris.

Hyphobacterium marinum sp. nov. and Hyphobacterium lacteum sp. nov., isolated from marine sediment.

Two bacterial strains, Y60-23T and HN-65T, were isolated from marine sediment samples collected from Xiaoshi Island, Weihai, and Dongzhai Harbour, Haikou, PR China, respectively. Based on the 16S rRNA gene sequences, strain Y60-23T exhibited 96.0% similarity to its most related type strain Hyphobacterium vulgare KCTC 52487T, while strain HN-65T exhibited 97.3% similarity to its most related type strain Hyphobacterium indicum 2ED5T. The 16S rRNA gene sequence similarity between the two strains was 95.8%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains Y60-23T and HN-65T belonged to the genus Hyphobacterium. Cells of strains Y60-23T and HN-65T were rod-shaped, Gram-stain-negative, aerobic, non-motile, prosthecate and multiplied by binary fission. The major cellular fatty acids (>10.0%) of strain Y60-23T were C18 : 1 ω7c and C17 : 0, while those of strain HN-65T were iso-C17 : 1 ω9c, iso-C17 : 0 and C18 : 1 ω7c. The major respiratory quinone in both strains was ubiquinone-10 (Q-10) and the major polar lipids were monoglycosyl diglyceride, sulfoquinovosyl diacylglycerol and glucuronopyranosyl diglyceride. The genomic DNA G+C contents of strains Y60-23T and HN-65T were 63.9 and 60.7 mol%, respectively. The average nucleotide identity value between the two strains was 72.1% and the DNA-DNA hybridization value was 18.4%, clearly distinguishing them from each other. According to the results of the phenotypic, chemotaxonomic, phylogenetic and genomic analyses, the two strains represented two novel species within the genus Hyphobacterium, for which the names Hyphobacterium marinum sp. nov. and Hyphobacterium lacteum sp. nov. were proposed with the type strains Y60-23T (=MCCC 1H01433T=KCTC 8172T) and HN-65T (=MCCC 1H01434T=KCTC 8169T), respectively.

Mechanism of Chinese sturgeon IFN-γ inhibition on Mycobacterium marinum (Acipenser sinensis).

IFN-γ plays a crucial role in both innate and adaptive immune responses and is a typical Th1 cytokine that promotes Th1 response and activates macrophages. When macrophages were incubated with IFN-γ, their phagocytosis ratio against Mycobacterium marinum increased significantly, as observed under fluorescence microscopy. The macrophages engulfed a large number of M. marinum. The proliferative ability of macrophages treated with IFN-γ was significantly weaker on the 4th and 7th day after phagocytosis and subsequent re-infection with marine chlamydia (P < 0.001). This suggests that IFN-γ enhances the phagocytosis and killing ability of macrophages against M. marinum. IFN-γ protein also significantly increased the production of reactive oxygen species (H2O2) and nitric oxide (NO) by macrophages. Additionally, the expression levels of toll-like receptor 2 (tlr2) and caspase 8 (casp8) were significantly higher in macrophages after IFN-γ incubation compared to direct infection after 12 h of M. marinum stimulation. Apoptosis was also observed to a higher degree in IFN-γ incubated macrophage. Moreover, mRNA expression of major histocompatibility complex (MHC) molecules produced by macrophages after IFN-γ incubation was significantly higher than direct infection. This indicates that IFN-γ enhances antigen presentation by upregulating MHC expression. It also upregulates tlr2 and casp8 expression through the TLR2 signaling pathway to induce apoptosis in macrophages. The pro-inflammatory cytokine showed an initial increase followed by a decline, suggesting that IFN-γ enhances the immune response of macrophages against M. marinum infection. On the other hand, the anti-inflammatory cytokine showed a delayed increase, significantly reducing the expression of pro-inflammatory cytokines. The expression of both cytokines balanced each other and together regulated the inflammatory reaction against M. marinum infection.

Manipulated C5aR1 over/down-expression associates with IL-6 expression during bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost.

Study on the Anti-Mycobacterium marinum Activity of a Series of Marine-Derived 14-Membered Resorcylic Acid Lactone Derivatives.

With the emergence of drug-resistant strains, the treatment of tuberculosis (TB) is becoming more difficult and there is an urgent need to find new anti-TB drugs. Mycobacterium marinum, as a model organism of Mycobacterium tuberculosis, can be used for the rapid and efficient screening of bioactive compounds. The 14-membered resorcylic acid lactones (RALs) have a wide range of bioactivities such as antibacterial, antifouling and antimalarial activity. In order to further study their bioactivities, we initially constructed a 14-membered RALs library, which contains 16 new derivatives. The anti-M. marinum activity was evaluated in vitro. Derivatives 12, 19, 20 and 22 exhibited promising activity with MIC90 values of 80, 90, 80 and 80 µM, respectively. The preliminary structure-activity relationships showed that the presence of a chlorine atom at C-5 was a key factor to improve activity. Further studies showed that 12 markedly inhibited the survival of M. marinum and significantly reduced the dosage of positive drugs isoniazid and rifampicin when combined with them. These results suggest that 12 is a bioactive compound capable of enhancing the potency of existing positive drugs, and its effective properties make it a very useful leads for future drug development in combating TB resistance.

Mycobacterium marinum Infection after Iguana Bite in Costa Rica.

Infections after reptile bites are uncommon, and microbial etiologies are not well defined. We describe a case of Mycobacterium marinum soft-tissue infection after an iguana bite in Costa Rica that was diagnosed through 16S rRNA sequencing and mycobacterial culture. This case informs providers of potential etiologies of infection after iguana bites.

Salinigranum marinum sp. nov. and Halohasta salina sp. nov., halophilic archaea isolated from sediment of a marine saltern and inland saline soil.

Two halophilic archaeal strains, ZS-10T and GSL13T, were isolated from the Zhoushan marine saltern in Zhejiang, and an inland saline soil from the Tarim Basin, Xinjiang, PR China, respectively. The cells of strain ZS-10T were pleomorphic while those of strain GSL13T were rod-shaped. Both of them stained Gram-negative and formed red-pigmented colonies on agar plates and their cells lysed in distilled water. The optimum growth of strain ZS-10T was observed at 40 °C, 3.4 M NaCl, 0.03 M MgCl2 and pH 7.5, while that of strain GSL13T was at 37 °C, 3.1 M NaCl, 0.5 M MgCl2 and pH 7.5. Phylogenetic and phylogenomic analyses indicated that these two strains were related to Salinigranum and Halohasta, respectively. Strains ZS-10T and GSL13T could be differentiated from the current members of Salinigranum and Halohasta based on the comparison of diverse phenotypic characteristics. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values among strain ZS-10T and current species of Salinigranum were 75.8-78.6 %, 80.6-81.9 % and 24.3-26.1 %, respectively. These values between strain GSL13T and current species of Halohasta were 78.4-80.8 %, 79.8-82.8% and 22.7-25.7 %, respectively, clearly below the threshold values for species demarcation. The polar lipids of strain ZS-10T were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulphated mannosyl glucosyl diether (S-DGD-1), while those of strain GSL13T were phosphatidic acid, PG, PGP-Me, phosphatidylglycerol sulphate and S-DGD-1. The polar lipid profile of strain GSL13T was identical to those of Halohasta, whereas strain ZS-10T did not contain the minor glycolipids detected in the current Salinigranum species. The phenotypic, phylogenetic and genome-based results suggested that strains ZS-10T (=CGMCC 1.12868T=JCM 30241T) and GSL13T (=CGMCC 1.15214T=JCM 30841T) represent two novel species, for which the names Salinigranum marinum sp. nov. and Halohasta salina sp. nov. are proposed.

Core single nucleotide polymorphism analysis reveals transmission of Mycobacterium marinum between animal and environmental sources in two aquaria.

Mycobacterium marinum is a slow-growing, photochromogenic nontuberculous mycobacterium, which can cause mycobacteriosis in various animals, including humans. Several cases of fish mycobacteriosis have been reported to date. Mycobacterium marinum has also been isolated from aquatic environmental sources such as water, sand, biofilms, and plants in the natural environments. Hence, we hypothesized that a wide variety of sources could be involved in the transmission of M. marinum. In this study, we tested this hypothesis by isolating M. marinum from various sources such as fish, invertebrates, seagrass, periphytons, biofilms, sand, and/or water in two aquaria in Japan and conducting a phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) using whole-genome sequences of the isolated strains. The analysis revealed that the strains from animal and environmental sources belonged to the same clusters. This molecular-based study epidemiologically confirmed that various sources, including fish, invertebrates, and environmental sources, could be involved in transmission of M. marinum in a closed-rearing environment. This is the first report where M. marinum was isolated from different sources, and various transmission routes were confirmed in actual cases, which provided essential information to improve the epidemiology of M. marinum.

Genital mycobacteriosis caused by Mycobacterium marinum detected in two captive sharks by peptide nucleic acid-fluorescence in situ hybridization.

Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)-infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M. marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M. marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat-shock protein gene (hsp65), and peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M. marinum. Because M. marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes.

Nitratireductor rhodophyticola sp. nov., isolated from marine red algae.

Two Gram-stain-negative, strictly aerobic bacteria, strains L1-7-SET and R6, isolated from marine red algae, were characterized. They shared 99.9 % 16S rRNA gene sequence similarity and a 100 % digital DNA-DNA hybridization (DDH) value, representing members of a single species. Cells of strains L1-7-SET and R6 were catalase- and oxidase-positive motile rods with a single polar flagellum. Strains L1-7-SET and R6 optimally grew at 30-35 °C, pH 7.0-8.0 and with 1.0-2.0 % (w/v) NaCl. Ubiquinone-10 was the sole isoprenoid quinone and C19 : 0 cyclo ω8c and summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c) were detected as the major cellular fatty acids. The DNA G+C contents of strains L1-7-SET and R6 were both 61.62 mol%. The polar lipids of strain L1-7-SET consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, an unidentified phospholipid and two unidentified polar lipids. Phylogenetic analyses based on 16S rRNA gene and 120 protein marker sequences revealed that strains L1-7-SET and R6 formed a phyletic lineage within the genus Nitratireductor and they were most closely related to Nitratireductor aquibiodomus NL21T and Nitratireductor kimnyeongensis KY 101T with both 98.8 % 16S rRNA gene sequence similarities. Digital DDH values between strain L1-7-SET and the type strains of N. aquibiodomus and N. kimnyeongensis were 60.3 and 29.5 %, respectively. The phenotypic, chemotaxonomic and molecular features support that strains L1-7-SET and R6 represents a novel species of the genus Nitratireductor, for which the name Nitratireductor rhodophyticola sp. nov. is proposed. The type strain is L1-7-SET (=KACC 19076T=KCTC 92231T=JCM 31802T).

Mycobacterium marinum cellulitis: A fishy etiology.

Skin and soft tissue infections account for 2% of emergency department visits annually, though more unusual causative bacteria associated with saltwater exposure may result in morbidity. Mycobacterium marinum represents a rare but important cause of cellulitis, which if untreated or improperly managed, can progress to dactylitis or osteomyelitis. This unusual diagnosis is made more challenging due to the prolonged incubation period of approximately 21 days, temporally separating the inoculation from the disease. Patients will present with a nodular rash in a sporotrichoid pattern. While doxycycline is one antibiotic providing saltwater coverage, M. marinum has variable sensitivities to anti-mycobacterial antibiotics, and thus biopsy helps confirm the diagnosis as well as provide sensitivities for treatment. Emergency clinicians should inquire about environmental risk factors when caring for patients with cellulitis, especially with atypical skin presentations, and consider M. marinum as a rare but important cellulitis etiology.

Disseminated Mycobacterium marinum in Human Immunodeficiency Virus Unmasked by Immune Reconstitution Inflammatory Syndrome.

Distinguishing disseminated Mycobacterium marinum from multifocal cutaneous disease in persons with human immunodeficiency virus/AIDS can present a diagnostic challenge, especially in the context of immune reconstitution inflammatory syndrome (IRIS). In this work, we demonstrate the utility of flow cytometry and whole genome sequencing (WGS) to diagnose disseminated M. marinum unmasked by IRIS following initiation of antiretroviral therapy. Flow cytometry demonstrated robust cytokine production by CD4 T cells in response to stimulation with M. marinum lysate. WGS of isolates from distinct lesions was consistent with clonal dissemination, supporting that preexisting disseminated M. marinum disease was uncovered by inflammatory manifestations, consistent with unmasking mycobacterial IRIS.

Structural analysis and functional study of phosphofructokinase B (PfkB) from Mycobacterium marinum.

Phosphofructokinase B (PfkB) belongs to the ribokinase family, which uses the phosphorylated sugar as substrate, and catalyzes fructose-6-phosphate into fructose-1,6-diphosphate. However, the structural basis of Mycobacterium marinum PfkB is not clear. Here, we found that the PfkB protein was monomeric in solution, which was different from most enzymes in this family. The crystal structure of PfkB protein from M. marinum was solved at a resolution of 2.21 Å. The PfkB structure consists of two domains, a major three-layered α/ß/α sandwich-like domain characteristic of the ribokinase-like superfamily, and a second domain composed of four-stranded ß sheets. Structural comparison analysis suggested that residues G236, A237, G238, and D239 could be critical for ATP catalysis and substrate binding of PfkB. Our current work provides new insights into understanding the mechanism of the glycolysis in M. marinum.

Pararhodonellum marinum gen. nov., sp. nov., isolated from deep-sea sediment.

A Gram-stain-negative, non-motile bacterial strain, designated SW124T, was isolated from a deep-sea sediment sample collected from the Indian Ocean. SW124T was aerobic and catalase-positive. The strain grew at 4-40 °C (optimum, 30 °C), at pH 5.0-8.6 (optimum, pH 7.0) and in the presence of 0.5-9.5 % (w/v) NaCl (optimum, 3.0%). The results of comparative analyses of the 16S rRNA gene sequence indicated that SW124T was grouped in the vicinity of the genus Rhodonellum in the family Cyclobacteriaceae. The quinone system contained menaquinone MK-7. The predominant cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). SW124T contained phosphatidylethanolamine (PE) as the major polar lipid. The genomic DNA G+C content of SW124T was 41.7 mol%. On the basis of its phenotypic characteristics and phylogenetic data, we propose that strain SW124T (=CGMCC 1.16171T=KCTC 82235T) represents a novel species of a novel genus, with the name Pararhodonellum marinum gen. nov., sp. nov.

Cysteiniphilum marinum sp. nov., isolated from coastal seawater.

Six aerobic Gram-negative bacteria were isolated from seawater in Guangdong Province, P.R. China. Cells were observed to be Gram-negative, aerobic, non-motile and non-spore forming. Growth of the designated type strain 19X3-30T occurred at a temperature range of 14-37 °C (optimum, 28 °C), a pH range of 6.0-8.0 (optimum, pH 7) and up to 7.5% NaCl (optimum, 1.5%; w/v), and was enhanced by CO2 and L-cysteine supplementation. The major polar lipids identified in strain 19X3-30T were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The principal cellular fatty acids profile showed the presence of anteiso-C15:0, anteiso-C17:0 and C18:0 (> 8% of total fatty acids), and the respiratory quinone was ubiquinone 8 (UQ-8). According to the analysis of 16S rRNA gene sequences, these strains represented a novel species within the family Fastidiosibacteraceae, sharing maximum similarities with Cysteiniphilum litorale DSM 101832T (96.6%) and Cysteiniphilum halobium DSM 103992T (95.3%). Phylogenetic dendrograms based on 16S rRNA gene and protein marker genes from the genomic sequences both indicated that the strains formed a monophyletic lineage closely linked to the genus Cysteiniphilum, which was also supported by the UPGMA dendrogram based on the MALDI-TOF MS profile. The genomic DNA G + C contents of six strains ranged from 38.0% to 38.1%. Based on different taxonomic genomic metrics, phylogeny and phenotypic features, we propose that the strains warrant the assignment to a novel species, for which the name Cysteiniphilum marinum sp. nov. is proposed. The type strain is 19X3-30T (= KCTC 82154T = CGMCC 1.18585T).