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.

Macrophage Epithelial Reprogramming Underlies Mycobacterial Granuloma Formation and Promotes Infection.

Mycobacterium tuberculosis infection in humans triggers formation of granulomas, which are tightly organized immune cell aggregates that are the central structure of tuberculosis. Infected and uninfected macrophages interdigitate, assuming an altered, flattened appearance. Although pathologists have described these changes for over a century, the molecular and cellular programs underlying this transition are unclear. Here, using the zebrafish-Mycobacterium marinum model, we found that mycobacterial granuloma formation is accompanied by macrophage induction of canonical epithelial molecules and structures. We identified fundamental macrophage reprogramming events that parallel E-cadherin-dependent mesenchymal-epithelial transitions. Macrophage-specific disruption of E-cadherin function resulted in disordered granuloma formation, enhanced immune cell access, decreased bacterial burden, and increased host survival, suggesting that the granuloma can also serve a bacteria-protective role. Granuloma macrophages in humans with tuberculosis were similarly transformed. Thus, during mycobacterial infection, granuloma macrophages are broadly reprogrammed by epithelial modules, and this reprogramming alters the trajectory of infection and the associated immune response.

Identification of Autophagy-Inhibiting Factors of Mycobacterium tuberculosis by High-Throughput Loss-of-Function Screening.

The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to progressive or latent infection. Autophagy is recognized as one component of host cell responses that has an essential role in innate and adaptive immunity to intracellular bacteria. Many microbes, including Mycobacterium tuberculosis, have evolved to evade or exploit autophagy, but the precise mechanisms and virulence factors are mostly unknown. Through a loss-of-function screening of an M. tuberculosis transposon mutant library, we identified 16 genes that contribute to autophagy inhibition, six of which encoded the PE/PPE protein family. Their expression in Mycobacterium smegmatis confirmed that these PE/PPE proteins inhibit autophagy and increase intracellular bacterial persistence or replication in infected cells. These effects were associated with increased mammalian target of rapamycin (mTOR) activity and also with decreased production of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). We also confirmed that the targeted deletion of the pe/ppe genes in M. tuberculosis resulted in enhanced autophagy and improved intracellular survival rates compared to those of wild-type bacteria in the infected macrophages. Differential expression of these PE/PPE proteins was observed in response to various stress conditions, suggesting that they may confer advantages to M. tuberculosis by modulating its interactions with host cells under various conditions. Our findings demonstrated that multiple M. tuberculosis PE/PPE proteins are involved in inhibiting autophagy during infection of host phagocytes and may provide strategic targets in developing therapeutics or vaccines against tuberculosis.

Multicenter Evaluation of the ePlex Respiratory Pathogen Panel for the Detection of Viral and Bacterial Respiratory Tract Pathogens in Nasopharyngeal Swabs.

The performance of the new ePlex Respiratory Pathogen (RP) panel (GenMark Diagnostics) for the simultaneous detection of 19 viruses (influenza A virus; influenza A H1 virus; influenza A 2009 H1 virus; influenza A H3 virus; influenza B virus; adenovirus; coronaviruses [HKU1, OC43, NL63, and 229E]; human rhinovirus/enterovirus; human metapneumovirus; parainfluenza viruses 1, 2, 3, and 4; and respiratory syncytial virus [RSV] [RSV subtype A and RSV subtype B]) and 2 bacteria (Mycoplasma pneumoniae and Chlamydia pneumoniae) was evaluated. Prospectively and retrospectively collected nasopharyngeal swab (NPS) specimens (n = 2,908) were evaluated by using the ePlex RP panel, with the bioMérieux/BioFire FilmArray Respiratory Panel (BioFire RP) as the comparator method. Discordance analysis was performed by using target-specific PCRs and bidirectional sequencing. The reproducibility of the assay was evaluated by using reproducibility panels comprised of 6 pathogens. The overall agreement between the ePlex RP and BioFire RP results was >95% for all targets. Positive percent agreement with the BioFire RP result for viruses ranged from 85.1% (95% confidence interval [CI], 80.2% to 88.9%) to 95.1% (95% CI, 89.0% to 97.9%), while negative percent agreement values ranged from 99.5% (95% CI, 99.1% to 99.7%) to 99.8% (95% CI, 99.5% to 99.9%). Additional testing of discordant targets (12%; 349/2,908) confirmed the results of ePlex RP for 38% (131/349) of samples tested. Reproducibility was 100% for all targets tested, with the exception of adenovirus, for which reproducibilities were 91.6% at low virus concentrations and 100% at moderate virus concentrations. The ePlex RP panel offers a new, rapid, and sensitive "sample-to-answer" multiplex panel for the detection of the most common viral and bacterial respiratory pathogens.

Mammalian target of Rapamycin inhibition and mycobacterial survival are uncoupled in murine macrophages.

BACKGROUND: Autophagy is a cellular response to intracellular pathogens including mycobacteria and is induced by the direct inhibitors of mammalian target of Rapamycin (mTOR), a major negative regulator of autophagy. Autophagy induction by mTOR inhibition (mTOR dependent autophagy), through chemical means or starvation, leads to mycobacterial killing in infected cells. However, previous work by our group has shown that mycobacterial infection of macrophages naturally induces both autophagy and mammalian target of Rapamycin (mTOR) activity (mTOR independent autophagy). In the current work, we further explore the relationship between mTOR activity and mycobacterial killing in macrophages. RESULTS: While low concentrations of the mTOR inhibitors, Rapamycin, Torin 1, and Torin 2, can effectively reduce or block mTOR activity in response to lipopolysaccharides (LPS) or mycobacteria, higher concentrations (10 uM) are required to observe Mycobacterium smegmatis killing. The growth of M. smegmatis was also inhibited by high concentrations of Rapamycin in LC3B and ATG5 deficient bone marrow derived macrophages, suggesting that non-autophagic mechanisms might contribute to killing at high doses. Since mycobacterial killing could be observed only at fairly high concentrations of the mTOR inhibitors, exceeding doses necessary to inhibit mTOR, we hypothesized that high doses of Rapamycin, the most commonly utilized mTOR inhibitor for inducing autophagic killing, may exert a direct bactericidal effect on the mycobacteria. Although a short-term treatment of mycobacteria with Rapamycin did not substantially affect mycobacterial growth, a long-term exposure to Rapamycin could impact mycobacterial growth in vitro in select species. CONCLUSIONS: This data, coupled with previous work from our laboratory, further indicates that autophagy induction by mTOR inhibition is an artificial means to increase mycobacterial killing and masks more relevant endogenous autophagic biochemistry that needs to be understood.

Candida auris rates in blood culture on the rise: results of US surveillance.

Candida auris is an emerging pathogen that poses a significant public health risk. Its multidrug resistance has led to high mortality, making rapid detection crucial for effective treatment and prevention of transmission. Recent data from the Centers for Disease Control and Prevention indicate a substantial increase in C. auris cases in the United States, with a 95% rise in 2021. To provide an update on the detection rates of C. auris, we analyzed blood culture results from a near real-time cloud-based surveillance network, BioFire Trend. From January 2021 to April 2023, 34 C. auris detections were observed. The analysis showed a notable increase in detections in 2023 compared to previous years. The detection rate in 2023 was higher in all four US Census Regions, except for the Northeast, where it remained constant. The findings emphasize the continuous rise in C. auris cases and highlight the importance of near real-time surveillance systems in monitoring this emerging pathogen.

Inhibition of apoptosis by Rv2456c through Nuclear factor-κB extends the survival of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen with several survival mechanisms aimed at subverting the host immune system. Apoptosis has been shown to be mycobactericidal, to activate CD8+ T cells, and to be modulated by mycobacterial proteins. Since few mycobacterial proteins have so far been directly implicated in the interactions between M. tuberculosis and host cell apoptosis, we screened M. tuberculosis H37Rv transposon mutants to identify mutants that fail to inhibit cell death (FID). One of these FID mutants, FID19, had a transposon insertion in Rv2456c and is important for survival in host cells. The lack of the protein resulted in enhanced caspase-3 mediated apoptosis, which is probably due to an inability to activate nuclear factor-κB. Additionally, FID19 infection enhanced polyfunctional CD8+ T cells and induced a higher frequency of interferon-γ secreting immune cells in a murine model. Taken together, our data suggest that Rv2456c is important for the survival of H37Rv by subduing the innate and ultimately adaptive immune responses of its host by preventing apoptosis of the infected cell. Better understanding of the host-mycobacterial interactions may be beneficial to develop novel drug targets and engineer more efficacious vaccine strains against tuberculosis.

CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.

Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.