Integrated single-cell transcriptome and T cell receptor profiling reveals defects of T cell exhaustion in pulmonary tuberculosis.

Tuberculosis-affected lungs with chronic inflammation harbor abundant immunosuppressive immune cells but the nature of such inflammation is unclear. Dysfunction in T cell exhaustion, while implicated in chronic inflammatory diseases, remains unexplored in tuberculosis. Given that immunotherapy targeting exhaustion checkpoints exacerbates tuberculosis, we speculate that T cell exhaustion is dysfunctional in tuberculosis. Using integrated single-cell RNA sequencing and T cell receptor profiling we reported defects in exhaustion responses within inflamed tuberculosis-affected lungs. Tuberculosis lungs demonstrated significantly reduced levels of exhausted CD8+ T cells and exhibited diminished expression of exhaustion-related transcripts among clonally expanded CD4+ and CD8+ T cells. Additionally, clonal expansion of CD4+ and CD8+ T cells bearing T cell receptors specific for CMV was observed. Expanded CD8+ T cells expressed the cytolytic marker GZMK. Hence, inflamed tuberculosis-affected lungs displayed dysfunction in T cell exhaustion. Our findings likely hold implications for understanding the reactivation of tuberculosis observed in patients undergoing immunotherapy targeting the exhaustion checkpoint.

Single-cell RNA-sequencing reveals heterogeneity and intercellular crosstalk in human tuberculosis lung.

Lung inflammation indicated by 18F-labeled fluorodeoxyglucose (FDG) in patients with tuberculosis is associated with disease severity and relapse risk upon treatment completion. We revealed the heterogeneity and intercellular crosstalk in lung tissues with 18F-FDG avidity and adjacent uninvolved tissues from 6 tuberculosis patients by single-cell RNA-sequencing. Tuberculous lungs had an influx of regulatory T cells (Treg), exhausted CD8 T cells, immunosuppressive myeloid cells, conventional DC, plasmacytoid DC, and neutrophils. Immune cells in inflamed lungs showed general up-regulation of ATP synthesis and interferon-mediated signaling. Immunosuppressive myeloid and Treg cells strongly displayed transcriptions of genes related to tuberculosis disease progression. Intensive crosstalk between IL4I1-expressing myeloid cells and Treg cells involving chemokines, costimulatory molecules, and immune checkpoints, some of which are specific in 18F-FDG-avid lungs, were found. Our analysis provides insights into the transcriptomic heterogeneity and cellular crosstalk in pulmonary tuberculosis and guides unveiling cellular and molecular targets for tuberculosis therapy.

Rapid GPR183-mediated recruitment of eosinophils to the lung after Mycobacterium tuberculosis infection.

Influx of eosinophils into the lungs is typically associated with type II responses during allergy and fungal and parasitic infections. However, we previously reported that eosinophils accumulate in lung lesions during type I inflammatory responses to Mycobacterium tuberculosis (Mtb) in humans, macaques, and mice, in which they support host resistance. Here we show eosinophils migrate into the lungs of macaques and mice as early as one week after Mtb exposure. In mice this influx is CCR3 independent and instead requires cell-intrinsic expression of the oxysterol receptor GPR183, which is highly expressed on human and macaque eosinophils. Murine eosinophils interact directly with bacilli-laden alveolar macrophages, which upregulate the oxysterol-synthesizing enzyme Ch25h, and eosinophil recruitment is impaired in Ch25h-deficient mice. Our findings show that eosinophils are among the earliest cells from circulation to sense and respond to Mtb infection of alveolar macrophages and reveal a role for GPR183 in the migration of eosinophils into lung tissue.

Negative Regulator Nlrc3-like Maintain the Balanced Innate Immune Response During Mycobacterial Infection in Zebrafish.

The NOD-like receptors (NLRs) have been shown to be involved in infection and autoinflammatory disease. Previously, we identified a zebrafish NLR, nlrc3-like, required for macrophage homeostasis in the brain under physiological conditions. Here, we found that a deficiency of nlrc3-like leads to decreased bacterial burden at a very early stage of Mycobacterium marinum infection, along with increased production of pro-inflammatory cytokines, such as il-1ß and tnf-α. Interestingly, myeloid-lineage specific overexpression of nlrc3-like achieved the opposite effects, suggesting that the impact of nlrc3-like on the host anti-mycobacterial response is mainly due to its expression in the innate immune system. Fluorescence-activated cell sorting (FACS) and subsequent gene expression analysis demonstrated that inflammasome activation-related genes were upregulated in the infected macrophages of nlrc3-like deficient embryos. By disrupting asc, encoding apoptosis-associated speck-like protein containing a CARD, a key component for inflammasome activation, the bacterial burden increased in asc and nlrc3-like double deficient embryos compared with nlrc3-like single deficient embryos, implying the involvement of inflammasome activation in infection control. We also found extensive neutrophil infiltration in the nlrc3-like deficient larvae during infection, which was associated with comparable bacterial burden but increased tissue damage and death at a later stage that could be alleviated by administration of dexamethasone. Our findings uncovered an important role of nlrc3-like in the negative regulation of macrophage inflammasome activation and neutrophil infiltration during mycobacterial infection. This highlights the importance of a balanced innate immune response during mycobacterial infection and provides a potential molecular basis to explain how anti-inflammatory drugs can improve treatment outcomes in TB patients whose infection is accompanied by a hyperinflammatory response.

Subinhibitory Concentrations of Antibiotics Exacerbate Staphylococcal Infection by Inducing Bacterial Virulence.

Antibiotics are widely used for the treatment of bacterial infections. However, injudicious use of antibiotics based on an empirical method may lead to the emergence of resistant strains. Despite appropriate administration of antibiotics, their concentrations may remain subinhibitory in the body, due to individual variations in tissue distribution and metabolism rates. This may promote bacterial virulence and complicate the treatment strategies. To investigate whether the administration of certain classes of antibiotics will induce bacterial virulence and worsen the infection under in vivo conditions. Different classes of antibiotics were tested in vitro for their ability to induce virulence in a methicillin-resistant S. aureus strain Mu3 and clinical isolates. Antibiotic-induced pathogenicity was assessed in vivo using mouse peritonitis and bacteremia models. In vitro, ß-lactam antibiotics and tetracyclines induced the expression of multiple surface-associated virulence factors as well as the secretion of toxins. In peritonitis and bacteremia models, mice infected with MRSA and treated with ampicillin, ceftazidime, or tetracycline showed enhanced bacterial pathogenicity. The release of induced virulence factors in vivo was confirmed in a histological examination. Subinhibitory concentrations of antibiotics belonging to ß-lactam and tetracycline aggravated infection by inducing staphylococcal virulence in vivo. Thus, when antibiotics are required, it is preferable to employ combination therapy and to initiate the appropriate treatment plan, following diagnosis. Our findings emphasize the risks associated with antibiotic-based therapy and underline the need for alternative therapeutic options. IMPORTANCE Antibiotics are widely applied to treat infectious diseases. Empirically treatment with incorrect antibiotics, or even correct antibiotics always falls into subinhibitory concentrations, due to dosing, distribution, or secretion. In this study, we have systematically evaluated in vitro virulence induction effect of antibiotics and in vivo exacerbated infection. The major highlight of this work is to prove the ß-lactam and tetracyclines antibiotics exacerbated disease is due to their induction effect on staphylococcal virulence. This phenomenon is common and suggests that if ß-lactam antibiotics remain the first line of defense during empirical therapy, we either need to increase patient reliability or the treatment approach may improve in the future when paired with anti-virulence drugs.

Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice.

Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. An influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, using complementary genetic models of eosinophil deficiency, we demonstrate that in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.

Long non-coding RNAs ENST00000429730.1 and MSTRG.93125.4 are associated with metabolic activity in tuberculosis lesions of sputum-negative tuberculosis patients.

Accurate diagnosis of complete inactivation of tuberculosis lesions is still a challenge with respect to sputum-negative tuberculosis. RNA-sequencing was conducted to uncover potential lncRNA indicators of metabolic activity in tuberculosis lesions. Lung tissues with high metabolic activity and low metabolic activity demonstrated by fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography were collected from five sputum-negative tuberculosis patients for RNA-sequencing. Differentially-expressed mRNAs and lncRNAs were identified. Their correlations were evaluated to construct lncRNA-mRNA co-expression network, in which lncRNAs and mRNAs with high degrees were confirmed by quantitative real-time PCR using samples collected from 11 patients. Prediction efficiencies of lncRNA indicators were assessed by receiver operating characteristic curve analysis. Bioinformatics analysis was performed for potential lncRNAs. 386 mRNAs and 44 lncRNAs were identified to be differentially expressed. Differentially-expressed mRNAs in lncRNA-mRNA co-expression network were significantly associated with fibrillar collagen, platelet-derived growth factor binding, and leukocyte migration involved in inflammatory response. Seven mRNAs (C1QB, CD68, CCL5, CCL19, MMP7, HLA-DMB, and CYBB) and two lncRNAs (ENST00000429730.1 and MSTRG.93125.4) were validated to be significantly up-regulated. The area under the curve of ENST00000429730.1 and MSTRG.93125.4 was 0.750 and 0.813, respectively. Two lncRNAs ENST00000429730.1 and MSTRG.93125.4 might be considered as potential indicators of metabolic activity in tuberculosis lesions for sputum-negative tuberculosis.

Increased Th17 activation and gut microbiota diversity are associated with pembrolizumab-triggered tuberculosis.

INTRODUCTION: A hypersensitivity response akin to immune reconstitution inflammatory syndrome (IRIS) has been proposed as a mechanism responsible for anti-PD-1 therapy-induced tuberculosis. IRIS is associated with enhanced activation of IL-17A-expressing CD4 + T cells (Th17). Gut microbiota is thought to be linked to pulmonary inflammation through the gut-lung axis. MATERIALS AND METHODS: We used ImmuCellAI to investigate the T cell population in lung cancer and tuberculosis samples. Then, we applied flow cytometry to monitor the expression levels of the Th17 cell activation marker CD38 in the peripheral blood of a patient experiencing adverse events, including tuberculosis, in response to pembrolizumab. The gut microbiome was examined by 16S rRNA sequencing to examine the alterations caused by pembrolizumab. RESULTS: The percentage of Th17 cells was increased in both lung cancer and tuberculosis. FACS analysis showed that pembrolizumab induced substantial CD38 expression in Th17 cells. The patient's fecal samples showed that the diversity of the gut microbiota was significantly increased in response to the pembrolizumab cycle. One enriched genus was Prevotella, which has previously been linked to lung inflammation and Th17 immune activation. DISCUSSION: The observed Th17 activation in our patient was consistent with a role of Th17-mediated IRIS in pembrolizumab-triggered tuberculosis. Pembrolizumab might trigger airway inflammation with a Th17 phenotype through microbiota interactions in the gut-lung axis.

Zinc transporter SLC39A7 relieves zinc deficiency to suppress alternative macrophage activation and impairment of phagocytosis.

Macrophages are key phagocytic cells and play an important role in eliminating external microorganisms and endogenous danger signals. Dysregulation in macrophage functions have been reported in patients with asthma. Zinc homeostasis is critical in maintaining macrophage functions. The solute carrier (SLC) protein SLC39A7, a Zn2+ importer, has recently been linked to asthma. However, the roles of SLC39A7 in macrophage phagocytosis are not well understood. Here we found that phagocytosis efficiency was significantly decreased in SLC39A7-knockdown THP-1 cells, however the phagocytosis capability could be reversed with zinc supplementation. SLC39A7 deficiency skewed macrophages towards alternative activation, as indicated by increased expression of M2 activation marker CD206 and decreased expression of M1 activation marker NOS2. Consistent to this result, SLC39A7-knockdown cells produced reduced amounts of proinflammatory cytokines TNF- and IL-6. Furthermore, the mRNA level of receptor Clec4e previously known to be involved in phagocytosis of BCG was significantly reduced in SLC39A7 knockdown cells. Importantly, all these defects due to SLC39A7 deficiency could be reversed by zinc supplementation. Thus, zinc transporter SLC39A7 provide support for phagocytosis and classical macrophage activation.

Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR1.

Activation of systemic acquired resistance in plants is associated with transcriptome reprogramming induced by the unstable coactivator NPR1. Immune-induced ubiquitination and proteasomal degradation of NPR1 are thought to facilitate continuous delivery of active NPR1 to target promoters, thereby maximising gene expression. Because of this potentially costly sacrificial process, we investigated if ubiquitination of NPR1 plays transcriptional roles prior to its proteasomal turnover. Here we show ubiquitination of NPR1 is a progressive event in which initial modification by a Cullin-RING E3 ligase promotes its chromatin association and expression of target genes. Only when polyubiquitination of NPR1 is enhanced by the E4 ligase, UBE4, it is targeted for proteasomal degradation. Conversely, ubiquitin ligase activities are opposed by UBP6/7, two proteasome-associated deubiquitinases that enhance NPR1 longevity. Thus, immune-induced transcriptome reprogramming requires sequential actions of E3 and E4 ligases balanced by opposing deubiquitinases that fine-tune activity of NPR1 without strict requirement for its sacrificial turnover.

Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria.

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG-encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.

The Role of ESX-1 in Mycobacterium tuberculosis Pathogenesis.

In this article, we have described several cellular pathological effects caused by the Mycobacterium tuberculosis ESX-1. The effects include induction of necrosis, NOD2 signaling, type I interferon production, and autophagy. We then attempted to suggest that these pathological effects are mediated by the cytosolic access of M. tuberculosis-derived materials as a result of the phagosome-disrupting activity of the major ESX-1 substrate ESAT-6. Such activity of ESAT-6 is most likely due to its pore-forming activity at the membrane. The amyloidogenic characteristic of ESAT-6 is reviewed here as a potential mechanism of membrane pore formation. In addition to ESAT-6, the ESX-1 substrate EspB interferes with membrane-mediated innate immune mechanisms such as efferocytosis and autophagy, most likely through its ability to bind phospholipids. Overall, the M. tuberculosis ESX-1 secretion system appears to be a specialized system for the deployment of host membrane-targeting proteins, whose primary function is to interrupt key steps in innate immune mechanisms against pathogens. Inhibitors that block the ESX-1 system or block host factors critical for ESX-1 toxicity have been identified and should represent attractive potential new antituberculosis drugs.

The Numerical Predominance and Large Transcriptome Differences of Neutrophils in Peripheral Blood Together Inevitably Account for a Reported Pulmonary Tuberculosis Signature.

Previous transcriptomic analysis revealed a 393-transcript signature (PTBsig), which is dominated by interferon inducible genes, in whole blood of pulmonary tuberculosis (PTB) patients. Comparisons with a limited set of interferon-driven genes among separated monocytes, CD4+ T cells, CD8+ T cells, and neutrophils indicated that the signature is due to changes in neutrophils, the overwhelmingly predominant cell type. By extending the analysis to the entire 393 transcripts of PTBsig and by switching the cell proportions between separated monocytes, CD4+ T cells, CD8+ T cells, and neutrophils, we create putative PTBsig for whole blood (pPTBsig) in which CD4+ or CD8+ T cells or monocytes predominated or in which the cell proportions were unchanged. These putative signatures are then compared to the actual reported PTBsig. We show that, because of their predominance in peripheral blood and their larger transcriptional responses, neutrophils were indeed almost exclusively responsible for PTBsig. We caution that the functional significance of changes in other cell types might escape notice in transcriptome analysis that is based upon whole blood.

Sendai Virus Mucosal Vaccination Establishes Lung-Resident Memory CD8 T Cell Immunity and Boosts BCG-Primed Protection against TB in Mice.

Accumulating evidence has shown the protective role of CD8+ T cells in vaccine-induced immunity against Mycobacterium tuberculosis (Mtb) despite controversy over their role in natural immunity. However, the current vaccine BCG is unable to induce sufficient CD8+ T cell responses, especially in the lung. Sendai virus, a respiratory RNA virus, is here engineered firstly as a novel recombinant anti-TB vaccine (SeV85AB) that encodes Mtb immuno-dominant antigens, Ag85A and Ag85B. A single mucosal vaccination elicited potent antigen-specific T cell responses and a degree of protection against Mtb challenge similar to the effect of BCG in mice. Depletion of CD8+ T cells abrogated the protective immunity afforded by SeV85AB vaccination. Interestingly, only SeV85AB vaccination induced high levels of lung-resident memory CD8+ T (TRM) cells, and this led to a rapid and strong recall of antigen-specific CD8+ T cell responses against Mtb challenge infection. Furthermore, when used in a BCG prime-SeV85AB boost strategy, SeV85AB vaccine significantly enhanced protection above that seen after BCG vaccination alone. Our findings suggest that CD8+ TRM cells that arise in lungs responding to this mucosal vaccination might help to protect against TB, and SeV85AB holds notable promise to improve BCG's protective efficacy in a prime-boost immunization regimen.

Association of time to prostate-specific antigen nadir and logarithm of prostate-specific antigen velocity after progression in metastatic prostate cancer with prior primary androgen deprivation therapy.

We investigated the association of time to prostate-specific antigen nadir (TTPN) and logarithm of prostate-specific antigen velocity after progression Log(PSAVAP) in metastatic prostate cancer with prior primary androgen deprivation therapy (ADT). All metastatic prostate cancer patients treated with primary ADT from 2000 to 2009 were reviewed. Patients who developed disease progression were included in the subsequent analyses. Patients were categorized into three groups according to their TTPN: TTPN of <3 months, 3-17 months, and >17 months. We compared the Log(PSAVAP) between the different TTPN groups using Mann-Whitney U-test and Kruskal-Wallis test. Further multiple linear regression analyses on Log(PSAVAP) were performed to adjust for other potential confounding factors. Among 419 patients who were treated with primary ADT, 306 patients developed disease progression with a median follow-up of 28 months. Longer TTPN was associated with lower Log(PSAVAP) (P = 0.008) within all subgroup analyses (TTPN of <3 vs 3-17 months, P= 0.020; TTPN of 3-17 vs >17 months, P= 0.009; and TTPN of <3 vs >17 months, P= 0.001). Upon multiple linear regression analyses, baseline PSA (regression coefficient 0.001, P= 0.045), PSA nadir (regression coefficient 0.002, P= 0.040), and TTPN (regression coefficient -0.030, P= 0.001) were the three factors that were significantly associated with Log(PSAVAP). In conclusion, a longer TTPN was associated with lower Log(PSAVAP) in metastatic prostate cancer patients following primary ADT. TTPN cut-offs at 3 months and 17 months appeared to have prognostic significance in predicting Log(PSAVAP). TTPN may serve as a good prognostic indicator in deciding the treatment strategy in patients with disease progression.

Survival outcomes of Chinese metastatic prostate cancer patients following primary androgen deprivation therapy in relation to prostate-specific antigen nadir level.

AIM: To evaluate the progression-free survival (PFS), cancer-specific survival (CSS) and overall survival (OS) of Chinese metastatic prostate cancer patients following primary androgen deprivation therapy (ADT) in relation to prostate-specific antigen (PSA) nadir level. METHODS: All Chinese prostate cancer patients with bone metastases who were treated with primary ADT from 2000 to 2009 were included. Patients' and disease characteristics were recorded. Patients were categorized into two PSA nadir groups (≤1.0 and >1.0 ng/mL). Associations of PSA nadir with PFS, CSS and OS were analyzed with Kaplan-Meier and Cox regression analyses. The survival outcomes of the two PSA nadir groups were presented. RESULTS: Four hundred nineteen patients were included in the study. PSA nadir appeared to be a good predictor for PFS (hazard ratio [HR] 1.86, 95% confidence interval [CI] 1.35-2.56, P < 0.001), CSS (HR 1.60, 95% CI 0.98-2.64, P = 0.063) and OS (HR 1.77, 95% CI 1.20-2.41, P < 0.001) upon multivariate Cox regression analyses. In the PSA nadir groups of ≤1.0 and >1.0 ng/mL, the median PFS were 15 and 10 months, and the 1-year PFS rates were 64% and 40%, respectively; the median CSS were 42 and 27 months, and the 5-year OS rates were 53% and 28%, respectively; and the median OS were 41 and 24 months, and the 5-year OS rates were 45% and 19%, respectively. CONCLUSIONS: Higher PSA nadir was associated with shorter PFS, CSS and OS in Chinese metastatic prostate cancer patients following primary ADT. The survival outcomes may serve as references in deciding the best treatment strategy in Chinese prostate cancer patients.

The mitosis-specific marker phosphohistone-H3 (PHH3) is an independent prognosticator in uterine smooth muscle tumours: an outcome-based study.

AIMS: Accurate mitosis counting, which is important in the diagnosis of uterine smooth muscle tumours (USMTs), is often difficult and subjective. The mitosis-specific immunohistochemical marker phosphohistone-H3 (PHH3) has been shown to be diagnostically useful, but its expression, in relation to outcome, has not been thoroughly investigated. The aim of this study is to evaluate PHH3 as a diagnostic and prognostic marker in USMTs. METHODS AND RESULTS: PHH3 expression was evaluated in 55 leiomyosarcomas (LMSs), 26 smooth muscle tumours of uncertain malignant potential (STUMPs), 18 leiomyomas with bizarre nuclei (LBN), and 12 leiomyomas (LMs). Scores were expressed as counts per 10 high-power fields (HPFs). Median follow-up durations of patients with LMS, STUMP, LBN and LM were, respectively, 39, 78, 65.5 and 49.5 months. Twenty-eight patients with LMSs (50.9%) died, and two (7.7%) patients with STUMPs experienced recurrence. The median PHH3 scores for LMSs were significantly higher than those for other categories of tumour. A score of ≥29/10 HPFs was also independently associated with a poor outcome. To test whether the PHH3 score could distinguish between benign USMTs with atypical histology and those that were clinically malignant, two biological groups were further delineated. Patients in group 1 (18 LBNs and 24 STUMPs) all had an uneventful outcome, whereas patients in group 2 (two recurrent STUMPs and 32 LMSs) all had a recurrence or tumour-related death. Median PHH3 scores for the two groups were, respectively, 2/10 HPFs and 27/10 HPFs. A PHH3 score of ≥7/10 HPFs was highly associated with malignancy. CONCLUSION: PHH3 is useful in evaluation of the biological behaviour of USMTs, and may serve as a prognostic indicator for LMSs.

Anterior perineal hernia after anterior exenteration.

Perineal hernia is a rare complication of anterior exenteration. We reported this complication after an anterior exenteration for bladder cancer with bleeding complication requiring packing and second-look laparotomy. Perineal approach is a simple and effective method for repair of perineal hernia.

Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence.

Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1-ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG-EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG-EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15-PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5-PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion.