Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors.

Introduction: As infection with Mycobacterium tuberculosis progresses, the bacilli experience various degrees of host stressors in the macrophage phagosome such as low pH, nutrient deprivation, or exposure to toxic agents, which promotes cell-to-cell phenotypic variation. This includes a physiologically viable but non- or slowly replicating persister subpopulation, which is characterised by a loss of growth on solid media, while remaining metabolically active. Persisters additionally evade the host immune response and macrophage antimicrobial processes by adapting their metabolic pathways to maintain survival and persistence in the host. Methods: A flow cytometry-based dual-fluorescent replication reporter assay, termed fluorescence dilution, provided a culture-independent method to characterize the single-cell replication dynamics of M. tuberculosis persisters following macrophage infection. Fluorescence dilution in combination with reference counting beads and a metabolic esterase reactive probe, calcein violet AM, provided an effective approach to enumerate and characterize the phenotypic heterogeneity within M. tuberculosis following macrophage infection. Results: Persister formation appeared dependent on the initial infection burden and intracellular bacterial burden. However, inhibition of phagocytosis by cytochalasin D treatment resulted in a significantly higher median percentage of persisters compared to inhibition of phagosome acidification by bafilomycin A1 treatment. Discussion: Our results suggest that different host factors differentially impact the intracellular bacterial burden, adaptive mechanisms and entry into persistence in macrophages.

Probing Dynamic Features of Phagosome Maturation in Macrophage using Au@MnOx @SiO2 Nanoparticles as pH-Sensitive Plasmonic Nanoprobes.

Phagosome maturation in macrophage is essential to the clearance of pathogenic materials in host defence but the dynamic features remain difficult to be measured in real time. Herein, we reported the multilayered Au@MnOx @SiO2 nanoparticle as a robust pH-sensitive plasmonic nanosensor for monitoring the dynamic acidification features over the phagosome maturation process in macrophage under darkfield microscopy. For this multilayered nanosensor, the gold nanoparticle core plays a role of signal reporter, the MnOx shell and the outmost SiO2 act as the sensing layer and the protecting layer, respectively. After subject to the acidic buffer solution, the MnOx layer in the multilayered nanoprobe could be decomposed rapidly, resulting in a remarkable spectral shift and color change under darkfield microscopy. We demonstrated this nanosensor for the investigation of single phagosome acidification dynamics by monitoring the color changes of nanoprobes after phagocytosis over time. The nanoprobes after phagocytosized in macrophage displayed a slight color change within the first hour and then cost several minutes to change from red to green in the next stage, indicating the phagosome undergoes a slow first and then fast acidification feature as well as a slow-to-fast acidification translation over the phagosome maturation process. Moreover, we validated that the slow-to-fast acidification translation was dependent on the activation of V-ATPase from the ATP depletion assay. We believed that this nanosensor is promising for studying the dynamic acidification features as well as disorders in phagosome maturation in phagocytic cells, which might provide valuable information for understanding the disease pathogenesis related to phagosome dysfunctions.

L-Arginine Enhances Intracellular Killing of Carbapenem-Resistant Klebsiella pneumoniae ST258 by Murine Neutrophils.

Carbapenem-resistant Klebsiella pneumoniae ST258 (CRKP-ST258) are a global concern due to their rapid dissemination, high lethality, antibiotic resistance and resistance to components of the immune response, such as neutrophils. Neutrophils are major host mediators, able to kill well-studied and antibiotic-sensitive laboratory reference strains of K. pneumoniae. However, CRKP-ST258 are able to evade neutrophil phagocytic killing, persisting longer in the host despite robust neutrophil recruitment. Here, we show that neutrophils are unable to clear a CRKP-ST258 isolate (KP35). Compared to the response elicited by a prototypic K. pneumoniae ATCC 43816 (KPPR1), the neutrophil intracellular response against KP35 is characterized by equivalent production of reactive oxygen species (ROS) and myeloperoxidase content, but impaired phagosomal acidification. Our results ruled out that this phenomenon is due to a phagocytosis defect, as we observed similar efficiency of phagocytosis by neutrophils infected with KP35 or KPPR1. Genomic analysis of the cps loci of KPPR1 and KP35 suggest that the capsule composition of KP35 explain the high phagocytosis efficiency by neutrophils. Consistent with other reports, we show that KP35 did not induce DNA release by neutrophils and KPPR1 only induced it at 3 h, when most of the bacteria have already been cleared. l-arginine metabolism has been identified as an important modulator of the host immune response and positively regulate T cells, macrophages and neutrophils in response to microbes. Our data show that l-arginine supplementation improved phagosome acidification, increased ROS production and enhanced nitric oxide consumption by neutrophils in response to KP35. The enhanced intracellular response observed after l-arginine supplementation ultimately improved KP35 clearance in vitro. KP35 was able to dysregulate the intracellular microbicidal machinery of neutrophils to survive in the intracellular environment. This process, however, can be reversed after l-arginine supplementation.

Determinants of Phagosomal pH During Host-Pathogen Interactions.

The ability of phagosomes to halt microbial growth is intimately linked to their ability to acidify their luminal pH. Establishment and maintenance of an acidic lumen requires precise co-ordination of H+ pumping and counter-ion permeation to offset the countervailing H+ leakage. Despite the best efforts of professional phagocytes, however, a number of specialized pathogens survive and even replicate inside phagosomes. In such instances, pathogens target the pH-regulatory machinery of the host cell in an effort to survive inside or escape from phagosomes. This review aims to describe how phagosomal pH is regulated during phagocytosis, why it varies in different types of professional phagocytes and the strategies developed by prototypical intracellular pathogens to manipulate phagosomal pH to survive, replicate, and eventually escape from the phagocyte.

The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification.

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

Differential Kinetics of Aspergillus nidulans and Aspergillus fumigatus Phagocytosis.

Invasive aspergillosis mainly occurs in immunocompromised patients and is commonly caused by Aspergillus fumigatus, while A.nidulans is rarely the causative agent. However, in chronic granulomatous disease (CGD) patients, A. nidulans is a frequent cause of invasive aspergillosis and is associated with higher mortality. Immune recognition of A. nidulans was compared to A. fumigatus to offer an insight into why A. nidulans infections are prevalent in CGD. Live cell imaging with J774A.1 macrophage-like cells and LC3-GFP-mCherry bone marrow-derived macrophages (BMDMs) revealed that phagocytosis of A. nidulans was slower compared to A. fumigatus. This difference could be attributed to slower migration of J774A.1 cells and a lower percentage of migrating BMDMs. In addition, delayed phagosome acidification and LC3-associated phagocytosis was observed with A. nidulans. Cytokine and oxidative burst measurements in human peripheral blood mononuclear cells revealed a lower oxidative burst upon challenge with A. nidulans. In contrast, A. nidulans induced significantly higher concentrations of cytokines. Collectively, our data demonstrate that A. nidulans is phagocytosed and processed at a slower rate compared to A. fumigatus, resulting in reduced fungal killing and increased germination of conidia. This slower rate of A. nidulans clearance may be permissive for overgrowth within certain immune settings.

Macrophage C-type lectin is essential for phagosome maturation and acidification during Escherichia coli-induced peritonitis.

Sepsis is a life-threatening condition caused by an uncontrolled response to bacterial infection. Impaired bactericidal activity in the host is directly associated with severe sepsis; however, the underlying regulatory mechanism(s) is largely unknown. Here, we show that MCL (macrophage C-type lectin) plays a crucial role in killing bacteria during Escherichia coli-induced peritonitis. MCL-deficient mice with E. coli-induced sepsis showed lower survival rates and reduced bacterial clearance when compared with control mice, despite similar levels of proinflammatory cytokine production. Although the ability of macrophages from MCL-deficient mice to kill bacteria was impaired, they showed normal phagocytic activity and production of reactive oxygen species. In addition, MCL-deficient macrophages showed defective phagosome maturation and phagosomal acidification after E. coli infection. Taken together, these results indicate that MCL plays an important role in host defense against E. coli infection by promoting phagosome maturation and acidification, thereby providing new insight into the role of MCL during pathogenesis of sepsis and offering new therapeutic options.

Mycobacterium tuberculosis Controls Phagosomal Acidification by Targeting CISH-Mediated Signaling.

Pathogens have evolved a range of mechanisms to counteract host defenses, notably to survive harsh acidic conditions in phagosomes. In the case of Mycobacterium tuberculosis, it has been shown that regulation of phagosome acidification could be achieved by interfering with the retention of the V-ATPase complexes at the vacuole. Here, we present evidence that M. tuberculosis resorts to yet another strategy to control phagosomal acidification, interfering with host suppressor of cytokine signaling (SOCS) protein functions. More precisely, we show that infection of macrophages with M. tuberculosis leads to granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion, inducing STAT5-mediated expression of cytokine-inducible SH2-containing protein (CISH), which selectively targets the V-ATPase catalytic subunit A for ubiquitination and degradation by the proteasome. Consistently, we show that inhibition of CISH expression leads to reduced replication of M. tuberculosis in macrophages. Our findings further broaden the molecular understanding of mechanisms deployed by bacteria to survive.