Polymyxin B and E (colistin) are the last resorts to treat multidrug-resistant Gram-negative pathogens. Pseudomonas aeruginosa is intrinsically resistant to a variety of antibiotics. The PhoP-PhoQ two-component regulatory system contributes to the resistance to polymyxins by regulating an arnBCADTEF-pmrE operon that encodes lipopolysaccharide modification enzymes. To identify additional PhoP-regulated genes that contribute to the tolerance to polymyxin B, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) assay and found novel PhoP binding sites on the chromosome. We further verified that PhoP directly controls the expression of PA14_46900, PA14_50740 and PA14_52340, and the operons of PA14_11970-PA14_11960 and PA14_52350-PA14_52370. Our results demonstrated that mutation of PA14_46900 increased the bacterial binding and susceptibility to polymyxin B. Meanwhile, mutation of PA14_11960 (papP), PA14_11970 (mpl), PA14_50740 (slyB), PA14_52350 (ppgS), and PA14_52370 (ppgH) reduced the bacterial survival rates and increased ethidium bromide influx under polymyxin B or Sodium dodecyl sulfate (SDS) treatment, indicating roles of these genes in maintaining membrane integrity in response to the stresses. By 1-N-phenylnaphthylamine (NPN) and propidium iodide (PI) staining assay, we found that papP and slyB are involved in maintaining outer membrane integrity, and mpl and ppgS-ppgH are involved in maintaining inner membrane integrity. Overall, our results reveal novel PhoP-PhoQ regulated genes that contribute to polymyxin B tolerance.

HIV-1 chronically infects host CD4⁺ T lymphocytes and further affects a variety of immune cells, including CD8⁺ T cells. In our previous study, by analyzing unbiased high-dimensional single-cell RNA-seq data (scRNA-seq), we found that the frequency of GZMK⁺CD8⁺ T cells expressing granzyme K (GZMK) was increased in people living with HIV-1 (PLWHs). However, the phenotypic and functional characteristics of these cells in chronic HIV-1 infection and their correlation with disease are not well understood. In this study, we conducted a comprehensive analysis of scRNA-seq and matched T-cell receptor repertoire (TCR) sequencing data to delve into the characterizations of GZMK⁺CD8⁺ T cells, which was further validated by flow cytometry. We observed heterogeneity within the GZMK⁺CD8⁺ T cells, which could be further subdivided into a GZMK⁺GZMB^- subset and a GZMK⁺GZMB⁺ subset, with the latter being significantly enriched in PLWHs. The GZMK⁺GZMB⁺ cells are a unique subset within CD8⁺ T cells, characterized by high proliferation, activation, inflammatory response, clone transition, etc., and are one of the differentiation endpoints by pseudotemporal analysis of CD8+αβ T cells. Despite being predominantly composed of effector memory T cells (Tem), similar to the GZMK⁺GZMB^- subset, the GZMK⁺GZMB⁺ subset exhibits differentiation at a later stage than the GZMK⁺GZMB^- subset. We also observed that the frequency/count of GZMK⁺GZMB⁺CD8⁺ T cells was negatively correlated with CD4/CD8 ratio, and positively correlated with HIV DNA, IP-10, and MIG levels in PLWHs. In vitro experiments demonstrate that GZMK can potentiate the stimulatory effects of lipopolysaccharide (LPS) on THP-1 macrophages via the TLR-4 pathway, significantly enhancing the secretion of IP-10, MIG, and MCP-1, as well as increasing the proportion of TNF-α⁺ cells. In conclusion, in PLWHs, GZMK⁺GZMB⁺CD8⁺ T cells are a highly reactive and inflammatory-inducing subset that may be associated with systemic inflammation.

People living with human immunodeficiency virus (PLWH) are a vulnerable population with a higher risk of severe coronavirus disease 2019 (COVID-19); therefore, vaccination is recommended as a priority. Data on viral reservoirs and immunologic outcomes for PLWH breakthrough infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently limited. In this study, we investigated the effects of SARS-CoV-2 breakthrough infection on hematological parameters, human immunodeficiency virus (HIV) reservoir size, and T-cell recovery in PLWH receiving antiretroviral therapy (ART) after SARS-CoV-2 booster vaccination. The results indicated that during breakthrough infection, booster vaccination with homologous and heterologous vaccines was safe in PLWH after receiving two doses of inactivated vaccination. The absolute CD4 counts decreased in the heterologous group, whereas the CD8 counts decreased in the homologous booster group after breakthrough infection in PLWH. Breakthrough infection increased HIV reservoirs and was associated with increased T-cell activation in PLWH who received virally suppressed ART and a 3-dose vaccination. According to our data, the breakthrough infection of SARS-CoV-2 may put PLWH at a greater risk for increased HIV reservoirs, even if these individuals were virally suppressed with ART after 3-dose SARS-CoV-2 vaccination.

Disruption of the gut microenvironment is a hallmark of HIV infection, where regulatory T cells (Tregs) play a critical role in maintaining gut homeostasis. However, the mechanisms by which gut Tregs contribute to immune reconstitution failure in HIV-infected individuals remain poorly understood. In this study, we employed single-cell RNA sequencing (scRNA-seq) to analyze gut Treg populations across three cohorts: eight immunological responders (IRs), three immunological non-responders (INRs), and four HIV-negative controls (NCs). Our findings revealed that INRs exhibit an increased proportion of gut Tregs but with significant functional impairments, including reduced suppressive capacity and heightened apoptotic activity. Notably, these Tregs underwent metabolic reprogramming in INRs, marked by an upregulation of glycolysis-related genes and a downregulation of the oxidative phosphorylation (OXPHOS) pathway. Additionally, both the abundance of short-chain fatty acid (SCFA)-producing bacteria and SCFA concentrations were reduced in INRs. In vitro SCFA supplementation restored Treg function by enhancing suppressive capacity, reducing early apoptosis, and rebalancing cellular energy metabolism from glycolysis to OXPHOS. These findings provide a comprehensive characterization of gut Treg dysfunction in INRs and underscore the therapeutic potential of targeting gut Tregs through microbiota and metabolite supplementation to improve immune reconstitution in HIV-infected individuals.