Cellular Microbiology

Pathogens have developed intricate strategies to overcome the hosts innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogen L. monocytogenes and host macrophages, a key event controlling the infection in vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from each other and non-cooperative, but a higher multiplicity of infection resulted in a reduced probability of replication. Using internalisation assays and conditional probabilities to mathematically describe the multi-stage invasion process, we demonstrate that the secreted Listeriolysin toxin (LLO) of the PrfA regulon regulates replication probability by compromising the ability to phagocytose bacteria. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encoding hly or actA genes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expression per se was not sufficient to increase the probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell PrfA-mediated cooperativity to regulate outcomes of host pathogen interactions. Key pointsO_LIL. monocytogenes invasion of innate immune macrophages results in heterogeneous infection outcomes at the single cell level C_LIO_LIFate of individual bacteria in the same host cell is independent from each other and non-cooperative C_LIO_LIBacterial populations coordinate host cell uptake via the rate of phagocytosis to reduce internalization at high MOI C_LIO_LIThe PrfA regulon system is necessary but not sufficient for L. monocytogenes replication, but population-level PrfA virulence regulates single cell outcome probability C_LI

Type I Interferons (IFNs) generally have a protective role during viral infections, but their function during bacterial infections is dependent on the bacterial species. Legionella pneumophila, Shigella sonnei and Mycobacterium tuberculosis can inhibit type I IFN signaling. Here we examined the role of type I IFN, specifically IFN{beta}, in the context of Salmonella enterica serovar Typhimurium (STm) macrophage infections and the capacity of STm to inhibit type I IFN signaling. We demonstrate that IFN{beta} has no effect on the intracellular growth of STm in infected bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice. STm infection inhibits IFN{beta} signaling but not IFN{gamma} signaling in a murine macrophage cell line. We show that this inhibition is independent of the type III and type VI secretion systems expressed by STm and is also independent of bacterial phagocytosis. The inhibition is Toll-like receptor 4 (TLR4)-dependent as the TLR4 ligand, lipopolysaccharide (LPS), alone is sufficient to inhibit IFN{beta}-mediated signaling and STm-infected, TLR4-deficient BMDMs do not exhibit inhibited IFN{beta} signaling. In summary, we show that macrophages exposed to STm have reduced IFN{beta} signaling via crosstalk with TLR4 signaling, and that IFN{beta} signaling does not affect cell autonomous host defense against STm.

The single flagellum of African trypanosomes is essential in multiple aspects of the parasite development. The FLAgellar Member 8 protein (FLAM8), localised to the tip of the flagellum in cultured insect forms, was identified as a marker of the locking event that controls flagellum length. Here, we investigated whether FLAM8 could also reflect the flagellum maturation state in other stages. We observed that FLAM8 distribution extended along the entire flagellar cytoskeleton in mammalian infective forms. Then, a rapid FLAM8 concentration to the distal tip occurs during differentiation into early insect forms, illustrating for the first time the remodeling of an existing flagellum in trypanosomes. In the tsetse cardia, FLAM8 further localizes to the entire length of the new flagellum during an asymmetric division. Strikingly, in parasites dividing in the tsetse midgut and in the salivary glands, the amount and distribution of FLAM8 in the new flagellum was seen to predict the daughter cell fate. We propose and discuss how FLAM8 could be considered as a meta-marker of the flagellum stage and maturation state in trypanosomes. Summary statementThe trypanosome protein FLAM8 displays a dynamic and stage-specific distribution during the entire parasite cycle, representing a novel marker of the flagellum stage and maturation state.

Intracellular pathogens rely on manipulating host endocytic pathways to ensure survival. Legionella and Chlamydia exploit host SNARE proteins, with Legionella cleaving syntaxin 17 (STX17) and Chlamydia interacting with VAMP8 and VAMP7. Similarly, Salmonella targets the hosts endosomal fusion machinery, using SPI effectors like SipC and SipA to interact with syntaxin 6 (STX6) and syntaxin 8 (STX8), respectively, maintaining its vacuolar niche. Recent evidence highlights syntaxin 7 (STX7), a Qa-SNARE involved in endo-lysosomal fusion, as a potential Salmonella target. BioID screening revealed STX7 interactions with SPI-2 effectors SifA and SopD2, suggesting a critical role in Salmonella pathogenesis. We investigated the role of STX7 in Salmonella-containing vacuole (SCV) biogenesis and pathogenesis in macrophages and epithelial cells. Our findings indicate that STX7 levels and localization differ between these cell types during infection, reflecting the distinct survival strategies of Salmonella. Live cell imaging showed that STX7 is recruited to SCVs at different infection stages, with significantly altered distribution in HeLa cells at the late stage of infection. STX7 knockdown resulted in reduced bacterial survival, which was rescued upon overexpression of STX7 in both HeLa and RAW264.7 cells, suggesting Salmonella hijacks STX7 to evade lysosomal fusion and secure nutrients for intracellular replication. These results underscore the essential role of STX7 in maintaining SCVs and facilitating Salmonella survival. Further, the temporal expression of STX7 adaptor/binding partners in macrophages showed dynamic interactions with STX7 facilitating Salmonella infection and survival in host cells. Together, our study highlights STX7 as a critical host factor exploited by Salmonella, providing insights into the molecular mechanisms underlying its pathogenesis in macrophages and epithelial cells. These findings may in form strategies for targeting host-pathogen interactions to combat Salmonella infections.

The single mitochondrion of the obligate intracellular parasite Toxoplasma gondii is highly dynamic. Toxoplasmas mitochondrion changes morphology as the parasite moves from the intracellular to the extracellular environment and during division. Toxoplasmas mitochondrial dynamic is dependent on an outer mitochondrion membrane-associated protein LMF1 and its interaction with IMC10, a protein localized at the inner membrane complex (IMC). In the absence of either LMF1 or IMC10, parasites have defective mitochondrial morphology and inheritance defects. As little is known about mitochondrial inheritance in Toxoplasma, we have used the LMF1/IMC10 tethering complex as an entry point to dissect the machinery behind this process. Using a yeast two-hybrid screen, we previously identified Myosin A (MyoA) as a putative interactor of LMF1. Although MyoA is known to be located at the parasites pellicle, we now show through ultrastructure expansion microscopy (U-ExM) that this protein accumulates around the mitochondrion in the late stages of parasite division. Parasites lacking MyoA show defective mitochondrial morphology and a delay in mitochondrion delivery to the daughter parasite buds during division, indicating that this protein is involved in organellar inheritance. Disruption of the parasites actin network also affects mitochondrion morphology. We also show that parasite-extracted mitochondrion vesicles interact with actin filaments. Interestingly, mitochondrion vesicles extracted out of parasites lacking LMF1 pulled down less actin, showing that LMF1 might be important for mitochondrion and actin interaction. Accordingly, we are showing for the first time that actin and Myosin A are important for Toxoplasma mitochondrial morphology and inheritance.

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-{beta} secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a {Delta}secA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the {Delta}secA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the {Delta}secA2 strain. Therefore, to better examine secreted IFN-{beta} levels when bacterial infection levels are equal across strains, we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-{beta} to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-{beta} from macrophages infected with the {Delta}secA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-{beta} response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.

Autophagy is a fundamental eukaryotic process that mediates clearance of unwanted molecules and facilitates nutrient release. The bacterial pathogen Legionella pneumophila establishes an intracellular niche within phagocytes by manipulating host cellular processes, such as autophagy. Effector proteins translocated by L. pneumophilas Dot/Icm type IV secretion system have been shown to suppress autophagy. However evidence suggests that overall inhibition of autophagy may be detrimental to the bacterium. As autophagy contributes to cellular homeostasis and nutrient acquisition, L. pneumophila may translocate effectors that promote autophagy for these benefits. Here, we show that effector protein Lpg2411 binds phosphatidylinositol-3-phosphate lipids and preferentially binds autophagosomes. Translocated Lpg2411 accumulates late during infection and co-localizes with the autophagy receptor p62 and ubiquitin. Furthermore, autophagy is inhibited to a greater extent in host cells infected with a mutant strain lacking Lpg2411 compared to those infected with wild-type L. pneumophila, indicating that Lpg2411 stimulates autophagy to support the bacteriums intracellular lifestyle. SummaryLegionella pneumophila translocates several effector proteins that inhibit autophagic processes. In this study, we find that the effector protein Lpg2411 targets autophagosomes during late stages of infection and promotes autophagy.

All alveolates including apicomplexa parasites contain an inner membrane complex (IMC) underneath the plasma membrane. The IMC is synthesized de novo during the daughter cell budding (endodyogeny) within the mother cell and serves as a crucial scaffold for supporting cytoskeletal structures and the glideosome machinery for the parasite locomotion. However, the mechanism(s) underlying the membrane biogenesis in the IMC are not well understood. Using clinically-relevant and globally-prevalent pathogenic protist model, Toxoplasma gondii, we identified the TgVAP-TgVPS13A-TgDAT1 complex bridging the IMC to the endoplasmic reticulum (ER) - the major site of phospholipid synthesis. Individual components of this complex play a crucial role in the IMC biogenesis, where the multi-modular TgVPS13A protein interacts with the lipid scramblase TgDAT1 in the IMC via its C-terminal VAB domain, and with the ER-resident TgVAP through its N-terminal region. DAT1 is recruited for the progeny formation sites during the early stages of budding. Conditional depletion of TgVPS13A, TgDAT1 or TgVAP results in collapse of the inner membrane complex, leading to parasite death, as visualized by endodyogeny-specific organelle markers. LactC2-GFP, a biosensor of phosphatidylserine and phosphatidylthreonine lipids made in the ER and enriched in the IMC, also mislocalizes upon protein depletion. In conclusion, we propose that TgVAP-TgVPS13A-TgDAT1 bridge the ER and IMC and mediate the inter-organelle transport of lipids, thus contributing to the organelle biogenesis and daughter budding in T. gondii. Author SummaryThe inner membrane complex (IMC) in apicomplexan parasites is essential for maintaining the structural stability of the parasite, as well as for its budding and motility. The IMC is composed of flattened vesicles, alveolins, and microtubules. However, the mechanism behind the biogenesis of these flattened vesicles remains unclear. In this study, we provide evidence that a protein complex formed by TgVAP, TgVPS13A, and TgDAT1 exists between the endoplasmic reticulum (ER) and the IMC. Phenotypic analysis indicates that the absence of any individual component of this protein complex disrupts the biogenesis of daughter IMCs, which in turn affects the budding of daughter parasites. We also demonstrated that TgDAT1 functions as a scramblase. Based on our findings, we propose a model in which the TgVAP-TgVPS13A-TgDAT1 complex mediates lipid transport from the ER to the nascent IMC, driving the expansion of the IMC membrane and the budding of daughter parasites in T. gondii. Furthermore, TgDAT1 may facilitate the transfer of lipids from the outer leaflet to the inner leaflet of the IMC sacs, promoting a balanced composition of the IMC membrane components.

All endo- and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the cells single flagellum. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, was proposed to affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of the hook complex component TbMORN1 resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form Trypanosoma brucei and was found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement and impaired access to the flagellar pocket membrane by surface-bound cargo, similar to depletion of TbMORN1. Unexpectedly, inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket. SummaryCharacterisation of the essential trypanosome protein TbSmee1 suggests that endocytosis is required for flagellar pocket access of surface-bound cargo.

We have previously shown that the slender form of Trypanosoma (T.) brucei is able to infect teneral tsetse flies, develop to the first fly form, which is the procyclic form, and complete the life cycle in the insect vector (Schuster et al., 2021). Further, analysis of the transmission index (TI; defined as the number of salivary gland infections relative to the number of midgut infections) revealed a higher TI for slender as compared to stumpy forms under laboratory conditions, which included the addition of N-acetyl-glucosamine (NAG) to the infective bloodmeal. These findings challenge the prevailing view of the life cycle, according to which only stumpy forms are considered infective to tsetse flies. Here, we show that slender trypanosomes can infect both male and female tsetse flies, irrespective of their teneral status, in the absence of supplements in the bloodmeal. Additionally, an RNA-sequencing time course was performed on both slender and stumpy cells during their transition into procyclic forms. This analysis revealed that slender and stumpy form trypanosomes remain transcriptionally distinct throughout differentiation into the procyclic form. Furthermore, while the protein associated with differentiation 1 (PAD1) remains essential for the transition, slender cells do not require expression of other hallmark stumpy form traits, such as cell cycle arrest or the shortening of their flagella or microtubule corset. Instead, slender trypanosomes are able to transition directly into procyclic forms. Taken together, these findings demonstrate that while slender cells of T. brucei follow distinct routes to become the procyclic form, they are capable of infecting both teneral and non-teneral tsetse flies, thereby contributing to the transmission and spread of these African parasites.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines the variable proportions of MSPDBL2 positivity in schizonts does not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous HA-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different to those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection.

Leishmania is a vacuolar pathogen that replicates within parasitophorous vacuoles inside host phagocytes. To promote its replication, Leishmania relies on a panoply of strategies to acquire macromolecules such as lipids from host macrophages. In this study, we have evaluated the role of VAPA, an endoplasmic reticulum-resident membrane protein involved in inter-organellar lipid transport, in macrophages infected with L. amazonensis. Following infection of bone marrow-derived macrophages with metacyclic L. amazonensis promastigotes, we observed that VAPA gradually associates with communal parasitophorous vacuoles. Knockdown of VAPA prevented the replication of L. amazonensis, which was accompanied by an impaired parasitophorous vacuole expansion. Using fluorescent ceramide, we established that VAPA is required for the transport of sphingolipids to the parasitophorous vacuoles and for its acquisition by L. amazonensis amastigotes. Proximity-ligation and immunoprecipitation assays revealed that L. amazonensis hijacks VAPA by disrupting its interactions with the lipid transfer proteins CERT and ORP1L. Finally, we found that VAPA is essential for the transfer of the Leishmania virulence glycolipid lipophosphoglycan from the parasitophorous vacuoles to the host cell endoplasmic reticulum. We propose that VAPA contributes to the ability of L. amazonensis to colonize macrophages by mediating bi-directional transfer of lipids essential for parasite replication and virulence between the parasitophorous vacuoles and the host cell endoplasmic reticulum. AUTHOR SUMMARYThe protozoan parasite Leishmania amazonensis replicates in macrophages, within communal parasitophorous vacuoles. To satisfy its various auxotrophies, this parasite must obtain macronutrients and metabolites from its host cell, including lipids. To salvage host sphingolipids, we obtained evidence that L. amazonensis exploits a macrophage nonvesicular lipid transport mechanism that requires the endoplasmic reticulum membrane protein VAPA. Moreover, we found that VAPA is also required for the transfer of the Leishmania virulence glycolipid lipophosphoglycan from the parasitophorous vacuole to the macrophage endoplasmic reticulum. The fact that VAPA is essential for L. amazonensis to colonize macrophages is consistent with the central role that VAPA plays in mediating bi-directional transfer of lipids and illustrates the importance of the host cell endoplasmic reticulum in this host-parasite interaction.

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in the vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. A report showed that Salmonella containing vacuole (SCV) harbors recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis is unclear. Upon knockdown of STX3, we have observed a reduction in bacterial proliferation and is restored upon the overexpression of STX3. Post infected live-cell imaging of cells showed STX3 localises to the SCV membranes and thus might help in fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found this interaction abrogated when we infected with SPI-2 encoded T3SS apparatus mutant (STM {Delta}ssaV) but not with SPI-1 encoded T3SS (STM{Delta} invC). These observations were also consistent in mice model of Salmonella infection. Together, these results shed a light on the effector molecules secreted through SPI-2 encoded by T3SS possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and maintenance the principle single bacterium per vacuole. SynopsisSalmonella Typhimurium infection in murine macrophage leads to upregulation of host Syntaxin 3 both at transcript and protein levels at late stage of infection. Syntaxin 3 cross-talk with Salmonella containing vacuoles (SCVs) is essential for establishment of replicative niche in host macrophages. The cross-talk between STX3 and SCVs is Salmonella pathogenicity island 2 (SPI-2) dependent and is consistent in mice model of Salmonella Typhimurium infection.

Toxoplasma gondii is an obligate, intracellular parasite capable of causing severe disease in warm-blooded animals. Infection of a cell produces a unique niche for the parasite named the parasitophorous vacuole (PV) initially composed of host plasma membrane invaginated during invasion. The PV and its membrane (PVM) are subsequently decorated with a variety of parasite proteins allowing the parasite to optimally grow in addition to manipulate host processes. Recently, we reported a proximity-labeling screen at the PVM-host interface and identified host ER-resident MOSPD2 as being enriched at this location. Here we extend these findings in several important respects. First, we show that the extent and pattern of host MOSPD2 association with the PVM differs dramatically in cells infected with different strains of Toxoplasma. Second, in cells infected with Type I RH strain, the MOSPD2 staining is mutually exclusive with regions of the PVM that associate with mitochondria. Third, immunoprecipitation and LC-MS/MS with epitope-tagged MOSPD2-expressing host cells reveals strong enrichment of several PVM-localized parasite proteins, although none appear to play an essential role in MOSPD2 association. Lastly, most MOSPD2 associating with the PVM is newly translated after infection of the cell and requires the major functional domains of MOSPD2, identified as the CRAL/TRIO domain and tail anchor, although these domains were not sufficient for PVM association. Collectively, these studies provide new insight into the molecular interactions involving MOSPD2 at the dynamic interface between the PVM and the host cytosol. ImportanceToxoplasma gondii is an intracellular pathogen that lives within a membranous vacuole inside of its host cell. This vacuole is decorated by a variety of parasite proteins that allow it to defend against host attack, acquire nutrients, and interact with the host cell. Recent work identified and validated host proteins enriched at this host-pathogen interface. Here, we follow up on one candidate named MOSPD2 shown to be enriched at the vacuolar membrane and describe it as having a dynamic interaction at this location depending on a variety of factors. Some of these include the presence of host mitochondria, intrinsic domains of the host protein, and whether translation is active. Importantly, we show that MOSPD2 enrichment at the vacuole membrane differs between strains indicating active involvement of the parasite with this phenotype. Altogether, these results shed light on the mechanism and role of protein associations in the host-pathogen interaction.

PDZ protein interacting specifically with Tc10 or PIST is a mammalian trans-Golgi resident protein that regulates subcellular sorting of plasma membrane receptors. PIST has recently been found to play an important role in regulating viral pathogenesis. Alteration in PIST expression is linked to the reprogramming of cell surface receptors which is crucial in determining herpes simplex virus1 infection. In this context, PIST is crucial in triggering autophagy via Beclin 1 -PI3KC3 pathway. However, there is complete lack in our knowledge on the role of this protein in any parasitic infection. Leishmania parasites infect their host macrophage cells via phagocytosis and their survival within the parasitophorous compartment has recently been found to be dependent on host autophagy by a yet to be identified mechanism. Using Leishmania major (L. major)-macrophage infection model system we, for the first-time report here that in infected macrophages Golgi resident PIST protein migrates towards the parasite containing compartment. We have also found that PIST associates with Beclin 1, however, not with LC3 within L. major parasite containing compartment of infected macrophages. Further, we performed genetic ablation of PIST by siRNA and observed that knockdown of macrophage PIST in turn helps in parasite replication. Contrary to this, overexpression of PIST in macrophages restricted the multiplication of L. major. Collectively, our study for the first time reveals that PIST is essential in regulating intracellular parasite, L. major infection within macrophage cells. Summary StatementMammalian PIST protein plays a crucial role in regulating cellular trafficking events. Here, we showed that PIST status is altered within Leishmania major parasite infected macrophages. Further, we confirmed that PIST is essential in restricting parasite growth. Additionally, a potential interacting axis between PIST and Beclin 1 is identified during infection.

The bloodstream form Trypanosoma brucei maintains essential mitochondrial membrane potential ({Delta}{Psi}m) through the reverse activity of FoF1-ATP synthase. The ATP that drives this activity is thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). We have shown that this carrier is the only carrier that can import ATP into the mitochondrial matrix to power the FoF1-ATPase. Contrary to expectations, its deletion has no effect on parasite growth, virulence and levels of {Delta}{Psi}m, suggesting that ATP is produced intramitochondrially by substrate phosphorylation pathways. Therefore, we knocked out the succinyl-CoA synthetase (SCoAS) gene, a key enzyme that produces ATP through substrate phosphorylation. Its absence resulted in changes in the metabolic landscape of the parasite, lower virulence, and reduced mitochondrial ATP content. This minimal mitochondrial ATP pool was maintained by AAC activity as evidenced by the 25- fold increase in sensitivity of the mutant parasites to AAC inhibitor carboxyatractyloside. Under nutrient-limited conditions, suppression of SCoAS expression by RNA interference negatively affected cell growth and levels of {Delta}{Psi}m. We concluded that the bloodstream mitochondrion is capable of generating ATP via substrate phosphorylation pathways, the importance of which depends on environmental conditions.

Autophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on mycobacterial phagosomes remain partially understood. In this study, we employed time-lapse confocal microscopy to investigate in real time the recruitment of LC3B (LC3), a key autophagy marker, to Mtb-containing vacuoles (MCVs) at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. The results reveal that approximately 70% of MCVs exhibited LC3 recruitment but was lost in about 40% of those MCVs. The LC3 recruitment to MCVs displayed a high variability in timing that was independent of the size of the MCV or the bacterial burden. Most notably, the LC3-positive MCVs did not acidify, indicating that LC3 recruitment does not necessarily lead to the formation of mature autophagolysosomes. In addition, interferon-gamma (IFN-{gamma}) pre-treatment did not affect LC3 recruitment frequency or autophagosome maturation, but increased the susceptibility of the macrophage to Mtb-induced cell death. Instead, LC3 recruitment and lysotracker staining were mutually exclusive events alternating on some MCVs multiple times showing a new reversible aspect of this autophagy response. It also suggested a role of autophagy in membrane repair of the MCV. Consistently, LC3 recruitment was strongly associated with galectin-3 and oxysterol-binding protein 1 staining, indicating a correlation with membrane damage and repair mechanisms. However, knockdown of ATG7 did not impact membrane repair, suggesting that autophagy is not directly involved in this process but is coregulated by the membrane damage of MCVs. In summary, our findings provide novel insights into the dynamic and variable nature of LC3 recruitment and autophagy to the MCVs over time during Mtb infection. Our data suggests that there is no major role of autophagy in cell autonomous defense against Mtb nor membrane repair of the MCV in human macrophages. However, the combined dynamics of LC3 recruitment and Lysoview staining emerged as promising markers for future research focused on directly investigating the damage and repair processes of phagosomal membranes.

Mycobacterium tuberculosis (Mtb) primarily infects human lung macrophages, which serve as its major replication niche. Mtb can manipulate host macrophage cell death pathways to its advantage by inhibiting apoptosis and inducing necrotic cell death. However, the specific necrotic cell death pathway activated in human macrophages after Mtb infection remains unclear. Here, we used the THP-1 cell line and primary human monocyte-derived macrophage (hMDM) to analyze multiple programmed cell death pathways during days 1-3 after Mtb infection. Confocal microscopic analysis demonstrates that Mtb-infected THP-1 cells or hMDMs rarely exhibited apoptosis. Immunoblotting shows that Mtb induces significant CASP3 and GSDME activation in THP-1 cells, but not in hMDMs. We show that Mtb, in THP-1 cells but not hMDM, induces a significant increase in GSDMD cleavage, a hallmark of pyroptosis. MLKL phosphorylation was not observed in THP-1 cells or hMDMs during Mtb infections, indicating an absence of necroptosis. No changes in ferroptosis markers such as GPX4 expression or lipid peroxidation levels were detected. Time-lapse live-cell imaging revealed no lysosomal membrane permeabilization prior to plasma membrane rupture (PMR). However, we observed DNA release from Mtb-infected THP-1 cells and hMDMs after PMR. The DNA released from THP-1 cells exhibits low levels of myeloperoxidase and histone H3 citrullination. High-resolution confocal imaging shows that Mtb is associated with the released DNA. We demonstrate that pyroptosis induction in THP-1 cells is dispensable for the DNA release and cell death induction. In conclusion, our results reveal that Mtb-triggered cell death in hMDMs bypasses canonical cell death pathways like apoptosis, pyroptosis, necroptosis, and ferroptosis. Instead, cell death in both THP-1 cells and hMDMs correlates with DNA release, potentially through a pathway similar to NETosis in neutrophils.

Trypanosoma brucei, the causal agent of Human and Animal African trypanosomiasis proliferates in the extracellular milieu of mammals. It acquires host macromolecular nutrients, by receptor mediated endocytosis. The best characterised cell surface receptor is for transferrin (TfR) and it has been reported to be preferentially localised in the flagellar pocket domain of the plasma membrane, the sole site of endocytosis. In this location the TfR may be inaccessible to adaptive immune system effectors. The T. brucei genome encodes [~]15 TfR variants, and here we compared two, the first attached to the plasma membrane by a single glycosylphosphatidylinositol (GPI)-anchor and the other by two. Transferrin uptake kinetics were similar and rapid for both. Unexpectedly, initial binding of transferrin occurred over the whole cell surface suggesting the TfR was not localised solely in the flagellar pocket. This localisation was confirmed by immunofluorescence assays and was independent of the number of GPI-anchors. Two other GPI-anchored receptors were investigated to determine whether localisation to the whole cell surface was a general property of GPI-anchored receptors. Haptoglobin-haemoglobin uptake assays and immunofluorescence localisation of complement factor H receptor showed both were also whole cell surface localised. The mechanisms by which trypanosome receptors are protected from antibody-mediated attack are more complex than hiding in a pocket.

Salmonella Typhimurium (STM) resides in a membrane-bound compartment called Salmonella containing vacuole (SCV) in several infected cell types. Within host cells, the division of bacteria and SCV are synchronous to maintain the single bacterium per vacuole. However, the mechanism regulating the synchronous fission and the machinery is not well understood. The fission of several intracellular organelles is regulated by the dynamic nature of the tubular endoplasmic reticulum (ER). In this study, we have evaluated the role of ER in controlling SCV fission. Interestingly, Salmonella-infected cells show the activation of unfolded protein response (UPR) with expanded ER tubules compared to the uninfected cells. Further, changing the expression of ER morphology regulators, such as reticulon-4a (Rtn4a) and CLIMP63, affected bacterial proliferation significantly, suggesting a potential role for tubular ER in facilitating the SCV division. Live-cell imaging analysis shows the marking of tubular ER precisely at the center of the majority of SCV division (78%) sites. We have investigated the role of SteA (a known Salmonella effector in modulating the membrane dynamics) in coordinating the SCV division. We observed that SteA resides on the SCV membranes and helps in making membrane contact sites between SCV and ER. Accordingly, the colocalization of ER with SCV enclosing SteA mutant Salmonella was significantly reduced compared to SCV-formed by wild-type Salmonella. Depletion of steA in Salmonella resulted in profound defects in SCV division, resulting in multiple bacteria residing in a single vacuole with defects in proliferation compared to the wild-type strain in epithelial cells. Also, during in vivo infection, the STM{Delta}steA mutant shows a defect in colonization in the spleen and liver and affects the initial survival rate of mice. Overall, this study suggests a coordinated role of bacterial effector SteA in promoting the ER contact sites with SCVs and thus regulating the successful division of SCV. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/592158v2_ufig1.gif" ALT="Figure 1000"> View larger version (72K): org.highwire.dtl.DTLVardef@21e83aorg.highwire.dtl.DTLVardef@157052org.highwire.dtl.DTLVardef@1815308org.highwire.dtl.DTLVardef@1724888_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG