Cellular Microbiology

Repressor-of-differentiation kinase 1 (RDK1) is one of two kinases expressed in bloodstream form Trypanosoma brucei parasites that were found to repress premature and spontaneous differentiation into the insect procyclic form. However, the effect of RDK1 RNAi was previously limited to the expression of a single surface coat protein, EP1 procyclin. Thus, there remains a significant gap in knowledge on the impact of RDK1 expression in bloodstream form T. brucei parasites. Here, we employ a systems biology approach and performed several proteomics analyses to identify RDK1 protein interactions and to determine the impact of loss of RDK1 expression on the bloodstream form proteome and phosphoproteome to uncover clues about potential mechanisms for RDK1 function. We found that RDK1 is dual localized to the cell membrane and the mitochondrial inner membrane with the kinase domain oriented towards the cytoplasm and mitochondrial inner membrane. Unexpectedly, the most enriched RDK1-proximal proteins were mitochondrial proteins. Furthermore, RDK1 depletion causes bloodstream form parasites to significantly upregulate many mitochondrial proteins and glycosomal proteins, several of which are upregulated in procyclic form parasites. Surprisingly, the mitochondrial phosphoproteome is largely unaffected by RDK1 depletion, while RDK1-dependent phosphoregulation is restricted to the cell membrane localization of RDK1. Lastly, we determined that RDK1 does not possess adenyl cyclase activity or alter intracellular cAMP levels; however, the dysregulated phosphoproteins correlate with functions in cyclic nucleotide signaling. In conclusion, RDK1 exhibits localization-specific kinase activity to regulate cyclic nucleotide signaling and mitochondrial proteomic maintenance in bloodstream form parasites. IMPORTANCETrypanosoma brucei is the unicellular parasite that causes African sleeping sickness and nagana disease in livestock across 36 sub-Saharan African countries. The parasite encounters different environmental niches as it is transmitted from an infected human to the tsetse fly vector as the fly takes a blood meal. T. brucei must sense environmental cues to initiate intracellular signaling pathways to promote effective differentiation and cellular remodeling from the mammalian bloodstream forms to the insect procyclic form. RDK1 is one of two kinases shown to repress premature differentiation to procyclic form, which would be detrimental for parasite survival in the human host. Therefore, it is essential to uncover mechanisms of RDK1 function to better understand how T. brucei maintains homeostasis in the human host and signals for effective cellular remodeling during parasite transmission.

Coxiella burnetii is a Gram-negative, obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Resident alveolar macrophages are the first target cells, but C. burnetii spreads to other cell types. While we have information about C. burnetii uptake and the establishment of the replication-competent phagolysosomal-like C. burnetii-containing vacuole (CCV), it is not well studied how C. burnetii exits its host cell. Here, we show that an infection with C. burnetii also triggers the activation of TFEB, a master regulator of autophagy and lysosomal development. The activation occurs in a time-dependent manner and depends on the size of the CCV. Importantly, TFEB activation during C. burnetii infection depend on MCOLN1, which channels Ca2+ across the lysosomal membrane into the cytosol. Knock-down of MCOLN1 resulted in reduced TFEB activation and smaller CCVs, while MCOLN1 activation boosted bacterial egress. Indeed, peripheral CCVs are positive for LAMP1/2 and release bacteria, without inducing host cell death. Importantly, LAMP1/2 and C. burnetii were stainable in non-permeabilized cells at sites of bacterial release, demonstrating fusion of the lysosome with the plasma membrane. Importantly, while replication of C. burnetii is not inhibited in cells lacking LAMP1/2, egress is impaired. Taken together, our data indicates that with increasing CCV size, TFEB is activated by the release of Ca2+ from lysosomes via the MCOLN1 channel, which in turn enables further CCV development and damage of the CCV membrane. This triggers lysosomal exocytosis and egress of C. burnetii without cell death induction.

1.In 2024, the World Health Organisation (WHO) classified Klebsiella pneumoniae as a maximum priority pathogen for the development of new alternatives to antibiotics. In this context, understanding the regulation of key virulence mechanisms is essential. Here, we investigated the role of the orphan quorum-sensing receptor SdiA in modulating virulence-associated processes during macrophage infection. Deletion of sdiA ({Delta}sdiA) significantly increased susceptibility to phagocytosis, as demonstrated using an amoeba predation model in which mutant strains formed larger clearance zones compared to wild-type bacteria. This phenotype was also observed in murine macrophages, where {Delta}sdiA strains exhibited increased adhesion (1.5 to 2.5-fold) and phagocytic uptake. Reduced uronic acid levels were also quantified in mutant strains, indirectly indicating a diminished capsule production, likely contributing to this enhanced phagocytosis. Despite enhanced uptake, {Delta}sdiA strains showed increased intracellular survival and replication rates within macrophages, leading to reduced host cell viability. This effect occurred despite loss of interbacterial killing capacity against E. coli, suggesting that enhanced intracellular fitness is not driven by classical antibacterial offensive mechanisms. Notably, mutant-infected macrophages displayed increased generation of reactive oxygen species (ROS), NF-{kappa}B expression, and pro-inflammatory cytokines (mCXCL10 and mTNF) production, indicating that macrophage defence mechanisms are not impaired during mutant infection. Overall, bacterial survival of {Delta}sdiA could result from overwhelming, rather than actively suppressing, host defences. Together, these findings identify SdiA as a negative regulator of phagocytosis and intracellular survival in K. pneumoniae and highlight a context-dependent role in virulence. This work provides new insights into the regulatory networks governing host-pathogen interactions and bacterial adaptation to the intracellular environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/725935v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d45bfdorg.highwire.dtl.DTLVardef@e3547forg.highwire.dtl.DTLVardef@c078f9org.highwire.dtl.DTLVardef@46408a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Loss of sdiA strongly affects phagocytosis, as mutant strains showed increasing adhesion (1.5 to 2.5-fold) and phagocytic uptake. Diminished capsule production could be contributing to this enhanced phagocytosis, as reduced uronic acid levels were also quantified in mutant strains. Despite being internalized at higher rates, mutants exhibited enhanced intracellular survival and replication, reducing macrophage viability. This fitness advantage occurred independently of classical offensive mechanisms, as evidenced by a lost ability to kill E. coli. Notably, mutant-infected macrophages mounted a stronger immune response, marked by elevated ROS, NF-{kappa}B expression, and pro-inflammatory cytokines production (mCXCL10 and mTNF). Together, these findings suggest that strains survive by overwhelming, rather than suppressing, host immune defences. Created with Biorender (https://www.biorender.com/). C_FIG HighlightsO_LISdiA deletion in K. pneumoniae increases susceptibility to phagocytosis. C_LIO_LIThe mutant strains exhibit reduced uronic acid levels, indicative of capsule production. C_LIO_LISdiA mutants show enhanced intracellular survival and higher macrophage death. C_LIO_LIMutant infected macrophages have higher NF-{kappa}B, TNF, and CXCL10 responses. C_LIO_LISdiA-deficient strains lose predatory capacity against E. coli. C_LI

Actin superfamily members are critical for the biology of eukaryotes and archaea. Actin-related proteins (Arps) are a subgroup within the actin superfamily and play essential roles in trafficking, replication and motility. The genome of the malaria parasite Plasmodium contains a set of Arps unique to apicomplexans, termed actin-like proteins (Alps). However, the importance and specific roles of many of these Alps in Plasmodium progression are not yet understood. Here, we determined the functional contribution of Plasmodium berghei Alp3 and Alp5a (recently relabelled as Arp3) by generation of knock-out (KO) lines and their subsequent characterisation across different life cycle stages. Deletion of either Alp did not affect blood stage growth, gametogenesis and ookinete gliding motility. However, deletion of Alp5a lead to smaller and fewer oocysts as well as severely impaired sporozoite formation. The Alp3KO line had highly reduced oocyst loads compared to wild-type parasites. This striking decrease was due to impaired ookinete penetration of the mosquito midgut epithelium. Our study shows that both Alp3 and Alp5a are indispensable for Plasmodium transmission at different steps of initial mosquito infection, provides insights into the role of specific unique members of the actin superfamily during parasite progression and the requirements for efficient midgut penetration.

Heterotrimeric kinesin 2 is the canonical motor protein for anterograde intraflagellar transport (IFT), driving movement of protein complexes towards the tip of cilia and flagella. Here, we show that all members of the Euglenozoa group lack genes for heterotrimeric kinesins and instead possess a variable number of genes for two homodimeric kinesins termed KIN2A and KIN2B. When expressed in vitro, both Trypanosoma brucei kinesins form homodimers and move processively along brain microtubules, KIN2A being faster than KIN2B. Studies in T. brucei and Leishmania mexicana show anterograde and retrograde IFT of both kinesins, with KIN2A travelling throughout the whole length of the flagellum, while KIN2B is concentrated at its base. In the proximal portion of the flagellum, most KIN2B molecules travel without IFT proteins, except for a few particles that are associated with IFT proteins and reach the tip. Surprisingly, the absence of KIN2A has mild effects on IFT and flagellum assembly, whereas KIN2B is essential for both. Investigation of trypanosome flagella deprived of KIN2B revealed that IFT proteins do not access these flagella but that KIN2A can still circulate. These results support a division-of-labour model where KIN2B is responsible for the import of IFT proteins while KIN2A is responsible for most of the anterograde transport.

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating immune responses and have emerged as potential biomarkers and therapeutic targets in complex diseases. Leishmaniasis is a neglected disease that compromises host immunity and is associated with challenging treatments regimens. Leishmania amazonensis (L. amazonensis), an intracellular protozoan parasite, causes cutaneous leishmaniasis by replicating inside mammalian macrophages to establish infection. In this context, miRNAs have emerged as vital post-transcriptional factors that regulate the inflammatory landscape during infection. In this study, we aimed to analyze the function of miR-721 in macrophages during L. amazonensis infection by integrating in silico miR-721 target prediction with RNAseq data from macrophages of two distinct mouse genotypes, resistant C57BL/6 and susceptible BALB/c. We found that miR-721 is induced in macrophages infected with L. amazonensis, but is not in LPS-stimulated macrophages, suggesting a TLR4-independent activation. Integrating miR-721 target prediction with comparative transcriptomic analyses in resistant C57BL/6 and susceptible BALB/c models revealed the TNF-IRF1 axis as a primary miR-721-associated regulatory network. Specifically, miR-721 is predicted to target the 3UTRs of Tnf and Irf1 to suppress the inflammatory response. Functional inhibition of miR-721 successfully restored Tnf and Irf1 expression and reduced the amastigote burden over 24 hours. Furthermore, we showed that the miR-721/TNF-IRF1 axis regulates downstream genes associated with macrophage response, such as Serpine1, Csf1, Cd69 and Maf. Our work demonstrated that Leishmania induces miR-721, which negatively modulates the TNF-IRF1 axis, thereby suppressing the immune response and favoring parasite persistence. While C57BL/6 macrophages exhibit a robust activation of the TNF-IRF1 network, promoting inflammatory response, BALB/c macrophage showed a breakdown of this network. This was associated with post-transcriptional suppression of inflammatory responses, thereby favoring parasite persistence. These findings link miR-721 to the establishment of macrophage polarization, providing relevant insights into the mechanisms of parasite subversion of the host immune response.

BackgroundStaphylococcus aureus (S. aureus) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. The mechanisms underlying these differences remain poorly understood. This study investigates how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. MethodsFour S. aureus isolates were analyzed for intracellular localization using transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. Transcriptional reprogramming of infected U937 monocytes was examined by mRNA sequencing. Infection outcomes were characterized and compared to A549 and SaOS-2 cell lines employing Luminex cytokine assays, flow cytometry and Western blot analysis to characterize host cell death mechanisms in both wild-type and TNF-R1 deficient backgrounds. ResultsAll S. aureus isolates localized to endolysosomal and cytosolic compartments but also peri and putatively intranuclearly, revealing an unexpected intracellular niche. In U937 monocytes, infection induced a conserved stress signature alongside isolatespecific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronicinfection isolate EDCC 5464. Cell death outcomes were likewise isolatedependent, involving intrinsic and extrinsic apoptosis, mitochondrial depolarization, and caspase-1 activation at distinct temporal dynamics. TNFR1 loss initially delayed but exacerbated late, isolate-independent cytotoxicity, identifying TNFR1 as a key regulator of U937 infection outcome. SaOS2 and A549 cell death was far less affected by isolate or TNF-R1 deficiency. ConclusionsThese results highlight the multilayered determinants governing intracellular S. aureus survival, non-canonical intracellular localization, and host cell susceptibility. The TNF/TNF-R1 axis is identified to critically determine regulated host defense during early infection stages in a tissue-specific manner. Together with distinct isolate-driven gene expression profiles, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies. Plain English summaryThe bacterium Staphylococcus aureus (S. aureus) often lives harmlessly in humans but can cause severe or recurrent infections when the skin barrier is broken or the immune system is weakened. A major reason for its persistence is its ability to hide inside human cells, where it is shielded from immune attacks and antibiotics. To effectively target such bacteria, it is crucial to understand that infections vary depending on both the bacterial strain and the infected cell type. Many reasons behind these differences are still puzzling. We explored how different types of S. aureus (collected from different disease types) change how human cells respond to infection. We focused on how the different strains influence the way immune cells adjust their gene activity during infection, and how a receptor called TNF-R1 is involved in managing cell death responses. Bacteria were found not only in compartments meant to destroy them but also near and even inside the cell nucleus, an unexpected location. All strains triggered a similar stress response but also distinct patterns influencing inflammation, metabolism, and cell survival. A strain linked to chronic infection caused weaker responses, suggesting greater stealth. Cells lacking TNF-R1 initially survived longer but later showed greater damage, indicating this receptors role in infection control. In lung and bone cells, these effects were less pronounced. Concludingly, S. aureus occupies unexpected niches inside human cells and uses varying survival strategies. TNF-R1 is a key regulator of host infection responses in the analyzed immune cells, highlighting that both bacterial diversity and host factors must be considered when developing targeted treatments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/723175v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1b4214org.highwire.dtl.DTLVardef@18f4ee6org.highwire.dtl.DTLVardef@1851742org.highwire.dtl.DTLVardef@ba0359_HPS_FORMAT_FIGEXP M_FIG Peri- and intranuclear localization early after S. aureus uptake across host cell lines, with isolate-specific modulation of host fates and a critical role for TNF-R1 to mediate regulated death responses of U937 cells. At 2 hpi, intracellular S. aureus not only localizes in (LAMP-1 decorated) membrane-enclosed compartments or directly in the cytosol, but within invaginations of the nuclear surface and intranuclearly with or without being surrounded by a vesicular membrane in U937wt, SaOS-2wt, and A549wt cells. At 4 hpi, S. aureus triggers differential gene expression in (A) U937wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates, that is muted for the chronic infection isolate EDCC 5464. Apoptotic cell death is induced to an isolate-dependent extent involving extrinsic initiator caspase-8, intrinsic initiator caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in the earlier stages of infection (6 hpi), which then barely increases (24 hpi) in U937wt cells. S. aureus-induced cell death and caspase activation is abolished in (B) U937{Delta}TNF-R1 at 6 hpi, but is significantly reinforced at 24 hpi with diminished isolate-specificity. Correspondingly, mitochondrial trans-membrane potential ({Delta}{Psi}m) is disrupted for all isolates upon TNF-R1 knockout, as well as caspase-1 activity, suggesting pyroptotic pathway activation at later stages of infection. (C) SaOS-2 wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of (D) A549wt cells with minimal caspase activation. Infection induces an isolate- and cell line-dependent cytokine release. Coloured arrows indicate the mean proportion of effector-positive cells ({uparrow} [~]20-40%, {uparrow} {uparrow} 40-60%, {uparrow} {uparrow} {uparrow} >60%) representing each S. aureus isolate. Grayed signaling arrows indicate the hypothesis by which TNF-R1 activation and internalization is required to kill lysosomal S. aureus via activation of anti-microbial enzymes and downstream regulated death pathway activation. Created with BioRender.com. C_FIG

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

Francisella tularensis is a highly virulent, Gram-negative bacterial pathogen that causes the zoonotic disease tularemia. F. tularensis infects a variety of host cells and replicates intracellularly while evading and interfering with host immune responses. The molecular mechanisms that facilitate the intracellular replication and virulence of F. tularensis are poorly understood. The Francisella genome contains a set of pil genes that code for the assembly of surface fibers termed type IV pili (T4P). T4P are major bacterial virulence determinants but the function of the pil system during F. tularensis infection and intracellular growth is unclear. T4P are closely related to the type II secretion pathway and the pil system of a related Francisella species, F. novicida, was shown to function in protein secretion as well as pilus assembly. To identify proteins secreted by F. tularensis, we analyzed the F. tularensis Live Vaccine Strain (LVS) using bio-orthogonal non-canonical amino acid tagging (BONCAT). Using BONCAT in conjunction with proteomics, we identified candidate proteins secreted by the wild-type LVS, as well as candidate proteins whose extracellular abundance decreased in the absence of the PilF ATPase or the PilE4 pilus subunit. Using epitope tagging of selected candidates, we validated T4P-mediated secretion of the ChiA and ChiD chitinases and the KatG catalase by the LVS. These results further our understanding of the pil system and protein secretion pathways in F. tularensis. IMPORTANCEFrancisella tularensis is a highly virulent Gram-negative bacterial pathogen and the causative agent of tularemia. F. tularensis lacks secretion systems utilized by other intracellular bacterial pathogens but contains pil genes that encode for type IV pili (T4P) and may also function in protein secretion. T4P are observed on the surface of all Francisella spp. but pil-mediated protein secretion has only been reported for F. novicida, which is not normally pathogenic in humans. In this study, we used bio-orthogonal non-canonical amino acid tagging to identify proteins secreted by F. tularensis, for which there is limited information. We demonstrate that the F. tularensis pil system is capable of protein secretion and validate T4P-medeated secretion of the ChiA and ChiD chitinases and the KatG catalase. These results will facilitate investigation of Francisella virulence mechanisms and may provide targets for therapeutic intervention.

The human malaria parasite Plasmodium falciparum (Pf) expresses ten different thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. PfTRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr256 and C-mannosylated at Trp250. We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two PfTRAP glyco-null mutant parasites, PfTRAP_T256A and PfTRAP_W250F, and assessed the fitness of these mutant parasites across the life cycle compared to the wild-type NF54 line as well as a PfTRAP knockout line. The PfTRAP glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. PfTRAP abundance in these mutants was significantly decreased despite normal transcript levels. Biophysical assays with recombinant proteins confirmed that glycosylation of the PfTRAP TSR stabilizes the domain and is likely required for its folding and secretion. These findings demonstrate that glycosylation of PfTRAPs TSR is critical for its proper expression and function, and underscore the importance of TSR glycosylation in the mosquito stage of the life cycle. IMPORTANCEMalaria is a mosquito-borne disease caused by Plasmodium parasites, of which P. falciparum is the deadliest. Plasmodium has ten proteins bearing thrombospondin type 1 repeats (TSRs), protein folds that aid cell-cell recognition and binding. Each of Plasmodiums ten TSR-bearing proteins is important for invading tissues in the mosquito vector and human host. TSRs are decorated with sugar molecules, a modification termed glycosylation. To better understand the importance of TSR glycosylation in Plasmodium, we investigated the P. falciparum protein TRAP, which is only expressed in mosquito-stage parasite forms called sporozoites. When PfTRAP was mutated to prevent glycosylation, abundance of the protein significantly decreased and parasites were unable to colonize the mosquito salivary glands. Furthermore, these mutant sporozoites were unable to move or to invade human liver cells. Our study reveals how TSR glycosylation can support the function of proteins that are required for parasite virulence.

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium that causes scrub typhus, a potentially life-threatening disease. To systematically identify host factors regulating early stages of infection, we performed a microscopy-based genome-wide siRNA screen in HeLa cells. This approach identified 2,989 genes grouped into 55 functional networks that modulate bacterial entry and intracellular translocation. In addition to confirming previously described pathways, including endocytosis and microtubule-dependent trafficking, the screen revealed an association between Ot infection and host cell cycle regulation. We found that Ot preferentially infects and/or replicates in host cells in the S and G2 phases, where intracellular bacterial accumulation is increased relative to G1. Early infection was associated with a shift in host cell cycle distribution, consistent with a delay in progression through S and G2 phases. Longitudinal analysis further showed that these cell cycle states support enhanced bacterial expansion. In parallel, infected cells exhibited reduced proliferation compared to uninfected cells, suggesting that Ot infection alters host cell division dynamics. Together, these findings support a model in which host cell cycle state influences susceptibility to Ot infection and intracellular growth. This work provides a systems-level map of host pathways involved in early infection and identifies cell cycle regulation as an important component of host-pathogen interactions in scrub typhus. Author SummaryScrub typhus is a potentially life-threatening disease caused by the bacterium Orientia tsutsugamushi, which can only survive and replicate inside human cells. Although some host factors involved in infection have been identified, many remain unknown. In this study, we used a large-scale screening approach to systematically identify human genes that influence the bacteriums ability to enter and move within host cells. Our analysis uncovered multiple pathways required for infection, including a role for the host cell cycle. We found that O. tsutsugamushi preferentially accumulates in cells during specific stages of the cell cycle, particularly when cells are preparing to divide. At the same time, infection slows host cell division, suggesting that the bacterium alters the cellular environment to support its own growth. These findings provide new insight into how O. tsutsugamushi interacts with human cells and identify potential host processes that could be targeted to limit infection.

The apicoplast of malaria parasites retains a reduced genome encoding a small set of genes with unknown functions. Among these genes is a putative ClpM chaperone, which unlike other apicoplast Clp-family members is not nuclear but plastid-encoded. In this study, we used ClpM as a model case to investigate evolutionary and molecular basis for plastid genome retention. Phylogenetic analyses across plastid-containing eukaryotes revealed that ClpM orthologues are broadly conserved and consistently plastid-encoded in all organisms with a red alga-derived plastid, irrespective of parasitism, photosynthesis or physiology. This broad phenomenon suggested gene-specific evolutionary constraints that were subsequently tested experimentally. To test whether clpM can be functionally expressed from the nucleus, we generated transgenic parasites carrying a nuclear ClpM copy fused to an apicoplast-targeting transit peptide. Unexpectedly, standard transgenesis resulted in transcriptional silencing, and we therefore forced transcription using integration into an endogenous essential locus. This led to robust clpM mRNA, however no detectable ClpM protein was observed. Multiple analyses ruled out apicoplast-dependent instability, ER-associated degradation, misfolding or membrane sequestration. Attempts to express clpM or other plastid-derived genes using endogenous sequences were found to be toxic, suggesting nucleotide-sequence incompatibility. In contrast, a transgene carrying a second copy of the nuclear ClpC ortholog was readily expressed. Comparative analysis of ClpM and ClpC domain architecture showed that their ATPase domains form distinct evolutionary clusters, suggesting conserved but functionally divergent roles. Subsequently, domain-swap experiments between ClpC and ClpM rescued partial expression and identified specific domains as contributors to the nuclear-expression barrier. Together, these findings demonstrate that clpM retention in the apicoplast genome is enforced by multilayered constraints involving evolutionary conservation, nucleotide-sequence incompatibility, transcriptional block and protein-intrinsic translational barriers. This work provides experimental evidence for mechanisms that restrict organelle-to-nucleus gene transfer and contribute to organelle genome retention.

The fungal pathobiont Candida albicans acquires heme from host proteins via a set of soluble and anchored extracellular CFEM-type hemophores that can capture the bound heme and exchange it, eventually delivering it to the cell membrane for endocytosis into the yeast cell. Yet the molecular mechanism by which the heme is transferred through this protein cascade across the cell envelope remains unclear. To address this mechanism, we developed a set a fusions of three C. albicans hemophores with fluorescent proteins. Fluorescence of these fusion proteins is strongly quenched when heme is bound to the hemophore moiety, enabling to measure heme transfer instantaneously. Kinetic analysis of the different transfer reactions reveals that heme transfer from the host protein to the CFEM hemophores and between the CFEM hemophores are governed by different regimes. Kinetics of heme transfer from hemoglobin or serum albumin to the CFEM hemophores is mainly first-order, suggesting that heme stochastically released from host proteins is captured by the hemophores. In contrast, transfer of heme between the hemophores is near second-order, consistent with a mechanism requiring protein-protein interactions. To confirm this, we show that CFEM hemophores can interact in homodimeric and heterodimeric complexes. Furthermore, while dimerization-defective mutants of the soluble hemophore Csa2 are proficient in heme binding and extraction, they are defective in heme transfer. This supports a model of heme transfer by direct interaction between the members of the fungal hemophore cascade. SIGNIFICANCEAcquisition of extracellular heme as iron or heme sources is common in microorganisms, and particularly prevalent among pathogenic organisms that must contend with an iron-poor host environment. To extract heme from host proteins, microorganisms deploy various systems that include extracellular soluble and cell-anchored hemophores. Here we describe a new approach for monitoring heme binding and transfer in real time, based on the development of fluorescent derivatives of fungal hemophores. These novel reagents open a new window on the study of a common virulence factor of microbial pathogens.

Plasmodium vivax (Pv) infections are developmentally asynchronous and often polyclonal, complicating interpretation of bulk parasite transcriptomes. Here, we analyzed paired in vivo and short-term ex vivo transcriptomes from Ethiopian clinical isolates using stage deconvolution and PvMSP1 haplotyping. Ex vivo maturation modestly increased inferred schizont representation while largely preserving the proportion of trophozoites and gametocytes. After adjustment for parasite stage composition, in vivo and ex vivo transcriptomes remained globally similar, with no genes significantly differentially expressed, indicating the absence of major culture-induced transcriptional response. In contrast, short-term culture reduced multiplicity of infection, contracted within-host haplotype diversity, and non-randomly depleted specific haplotypes, consistent with a clonal bottleneck. In a subset of low-complexity infections, residual expression patterns were clustered by dominant haplotype, suggesting genotype-associated transcriptional heterogeneity independent of developmental stage. Together, these findings indicate that short-term ex vivo culture enriches late asexual stages and selectively filters clones rather than inducing a common transcriptional program. These results shows that ex vivo cultures are reliable way to study gene expression, especially for late stages. However, these needs explicitly model developmental composition and infection complexity when interpreting Pv transcriptomes from natural infections Author summaryMalaria caused by Plasmodium vivax is difficult to study because this parasite cannot yet be grown continuously in the laboratory and infections in patients often contain parasites at different developmental stages and multiple parasite lineages at the same time. In this study, we wanted to understand how much of the parasite gene-expression signal reflects true biological differences, and how much is explained by parasite development or changes that occur during short-term laboratory maturation. We compared parasites collected directly from patients in Ethiopia with matched parasite matured briefly outside the body. We found that short-term culture mainly increased the proportion of later-stage parasites, but after accounting for developmental stage, the overall gene-expression patterns remained very similar. However, culture reduced the diversity of parasite lineages within infections, suggesting that some parasite lineages survive better than others under laboratory conditions. Our findings highlight that natural Pv infections are complex mixtures of parasite stages and lineages. Accounting for this complexity will improve how researchers interpret parasite gene-expression studies and design future studies of parasite invasion, transmission, and survival.

Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus burden at 3 h post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support early infection by B. abortus and B. ovis without impairing general phagocytosis. Gene set enrichment analysis further revealed positive regulators of mTORC1 signaling as key host factors; this result was validated through targeted disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Thus, the AKT-Ragulator-mTORC1 signaling axis contributes to the establishment of a permissive intracellular niche during early Brucella infection. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that support early Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.

Respiratory viral-bacterial co-infections cause severe disease across species, yet the molecular mechanisms underlying enhanced pathogenesis remain poorly understood. This study characterised H3N8 equine influenza A virus (IAV) and Streptococcus equi subspecies zooepidemicus (SEZ) co-infections using complementary ultrastructural and transcriptomic approaches. Transmission electron microscopy demonstrated direct physical binding between spherical (A/equine/Miami/63) and filamentous (A/equine/Sussex/89 and A/equine/Newmarket/5/2003) IAV isolates and SEZ, including when SEZ was heat-inactivated ({theta}SEZ). Lectin staining revealed that SEZ expresses predominantly 2,3-linked sialic acids, the receptor for equine IAV. However, virus-bacteria binding persisted despite neuraminidase treatment. Scanning electron microscopy quantification demonstrated that viral pre-infection significantly enhanced bacterial adherence to cells of the DH82 canine macrophage-like cell line (2-fold increase, p<0.01) but not ExtEqFL (equine lung-derived) cells, revealing cell-type-specific enhancement. RNA-sequencing analysis showed that bacterial infection drove most transcriptional changes during co-infection with little difference in the number of differentially expressed genes (DEGs) between infection with SEZ alone (146 DEGS) or after pre-infection with either A/equine/Sussex/89 (166 DEGS) or A/equine/Newmarket/5/2003 (149 DEGS). Validation of upregulation of selected cytokines by RT-qPCR and ELISA demonstrated that SEZ infection drives dramatic cytokine upregulation compared to mock or {theta}SEZ controls. Viral pre-infection did not alter the SEZ-induced pro-inflammatory cytokine responses (IL-6, IL-8, TNF-) but significantly reduced IFN-{beta} expression compared to SEZ infection alone. These findings suggest that direct virus-bacteria physical interactions may drive cell-type-specific enhancement of bacterial colonisation, fundamentally advancing our understanding of respiratory co-infection pathogenesis.

Flagella are large transenvelope nanomachines but how they transit the peptidoglycan in Gram positive bacteria is poorly understood. A recent model suggested that flagellar basal bodies diffuse in the membrane and become captured at locations in the peptidoglycan with a pore diameter that could accommodate the axle-like flagellar rod. Mutation of penicillin binding protein 1 (PBP1/PonA), a cell wall repair protein thought to decrease peptidoglycan pore frequency and/or size, resulted in a severe growth defect and cell lysis in the ancestral strain of Bacillus subtilis that was dependent on flagellar synthesis. Genetic analysis indicated that toxicity was due to completion of the flagellar hook, which activated the flagellar sigma factor SigD. SigD, in turn, activated a suite of peptidoglycan hydrolases that caused cellular lysis when PBP1 was absent. In addition, mutations that resulted in high levels of the stress response factor Spx could lessen the toxicity, while PBPX, a putative teichoic acid D-alanylase, was required for autolysis. In sum our results indicate that flagellar synthesis, not normally associated with cell viability, causes cell wall stress and under some conditions, cell death. Moreover, our work indicates that cost of envelope integrity by flagellar synthesis may be underappreciated due to strain domestication, and suggests that specialized systems may compensate for the cost of assembly of transenvelope machines in general. SIGNIFICANCEBacteria assemble nanomachines through the cell envelope but how the machines transit the peptidoglycan is poorly understood. Here we find that assembly of trans-envelope flagella results in cell lysis of Bacillus subtilis when the peptidoglycan repair protein PBP1 is absent. Lysis was due to multiple peptidoglycan lyases expressed as a consequence of flagellar assembly, and lytic activity required another PBP homolog, PBPX. Our work indicates that flagella, not normally thought to impact cell viability, can be lethal at the level of cell envelope integrity.

Plasmodium falciparum malaria parasites harbor an essential plastid organelle, called the apicoplast, which produces key metabolites required for organelle function and parasite viability. Apicoplast functions depend on iron and other metals, but the membrane transporters that mediate metal import into this organelle have been challenging to identify. Tetracycline antibiotics, including doxycycline, specifically target the apicoplast and can exhibit metal-dependent activity. Using tetracycline-affinity proteomics, we identified a doxycycline-interacting, uncharacterized transmembrane protein (UCT) targeted to the apicoplast periphery but not proteolytically processed. Although lacking sequence similarity to proteins of known function, UCT has a predicted structure with high similarity to pentameric CorA-family metal transporters that mediate metal uptake in other organisms. Functional tests revealed that UCT is dispensable for blood-stage asexual parasites, suggesting that the apicoplast has evolved redundant mechanisms for metal uptake. UCT knockdown in gametocytes, however, impairs the development of sexual parasites, which are critical for mosquito transmission. Our study identifies an apicoplast membrane protein with localization and structural properties that predict a role in metal transport into this key organelle. This discovery can provide a biochemical springboard to unravel broader apicoplast mechanisms of metal uptake across multiple stages of parasite development, including mosquito-stage parasites that display heightened UCT expression.

Mycobacterium tuberculosis (Mtb) cultured in minimal medium at acidic pH arrests its growth when provided specific single carbon sources, including glycerol, propionate, and lactate, a phenomenon we refer to as acid growth arrest. To define mechanisms of acid growth arrest on lactate, transposon mutants that suppress growth arrest were selected. Four mutants had insertions in phoT and one had an insertion in pstC2, both components of a phosphate ABC transporter. Mtb grows in minimal media supplemented with lactate at acidic pH when phosphate is depleted, showing that Mtb growth arrest on lactate is dependent on phosphate. The combination of lactate and phosphate at acidic pH causes cytoplasmic acidification below pH 6.7 in wild type Mtb, but a phoT::Tn mutant maintains a cytoplasmic pH of >7.2. Membrane potential in wild type Mtb is slightly decreased by lactate in a dose-dependent manner but is higher in the phoT::Tn mutant. Thus, acidic pH, phosphate, and lactate act together to dissipate proton motive force (PMF), a stress that is associated with acid growth arrest. Transcriptional profiling further supports that lactate causes PMF stress including induction of electron transport chain genes. The phoT::Tn mutant grown in lactate at acidic pH upregulates the senX3/regX3 regulon and using a regX3 mutant, we demonstrate that growth on lactate at low phosphate requires regX3. We propose a model where 1) the combined impact of acidic pH, lactate, and phosphate drives cytoplasmic pH acidification and decreased PMF, thus promoting acid growth arrest, and 2) low phosphate or a mutated phosphate transporter causes upregulation of senX3-regX3, which may induce ESX-5 and PPE/PE-based import mechanisms, thereby altering the mycomembrane or nutrient uptake in a manner that promotes growth on lactate at acidic pH. ImportanceMycobacterium tuberculosis (Mtb) grows well on lactate as a sole carbon source at neutral pH, but not at acidic pH. This study sought to understand why there is a pH-dependent growth restriction on lactate. A genetic selection for mutants that can grow on lactate at acidic pH identified mutants defective in phosphate transport. We found that limiting phosphate through depleting extracellular availability or inactivating a phosphate transporter promotes growth on lactate at acidic pH, and that this growth is dependent on the phosphate responsive two-component regulatory system SenX3-RegX3. Furthermore, we show that lactate, phosphate, and acidic pH combine to cause cytoplasmic pH acidification, a metabolic stress that is associated with acid growth arrest on lactate.

Signaling pathways often share components yet produce highly specialized biological responses. How signaling specificity is achieved between pathways utilizing common components is a fundamental question. In budding yeast, the same transmembrane mucin, Msb2, regulates two Mitogen-Activated Protein Kinase (MAPK) pathways controlling filamentous growth (fMAPK) and the response to osmotic stress (HOG). How this shared sensor distinguishes between stimuli and regulates different pathways is not clear. Using structure-guided analysis, we identified a conserved SEA (Sea urchin sperm protein, Enterokinase, Agrin) domain in fungal mucins and found that mutations disrupting protein folding selectively impair one pathway (fMAPK) but were tolerated by another (HOG). Mechanistically, these differences revealed distinct modes of signal transmission. The fMAPK pathway required an intact SEA domain and the cytosolic tail, consistent with a cis signaling mechanism that required structural coupling across the membrane. In contrast, the HOG pathway functioned independently of the cytosolic tail and tolerated misfolded SEA domain variants, consistent with trans signaling mediated by extracellular domains of interacting partners. The HOG pathway may detect misfolding as part of its sensing mechanism, as stressors that induce protein misfolding required Msb2 for survival. This work reveals how differential tolerance to protein deformation confers signaling specificity and identifies sensor deformation as a general feature of mechanosensory pathways that respond to environmental stress. HIGHLIGHTSO_LISignaling pathways differ in tolerance to misfolding of a sensory domain C_LIO_LIMisfolded SEA domains retain function in a stress pathway (HOG) pathway but not a cell differentiation pathway (fMAPK) O_LIMisfolded SEA domain variants showed altered protein levels, mis-localization in the secretory pathway, and turnover by ERAD C_LIO_LINon-functional variants lacked residues that stabilize the structure through intramolecular bonds C_LI C_LIO_LIDifferential tolerance for misfolding revealed distinct modes of signaling O_LITrans signaling predominated in the HOG pathway and did not require proper SEA domain folding or the mucin cytosolic tail O_LIA dominant hyperactive variant next to the SEA domain revealed basal interactions with the CR domain of tetraspanin C_LIO_LIAlphaFold modeling showed distinct interactions occur between the SEA domain and tetraspanin in the basal and activated states C_LI C_LIO_LICis signaling predominated in the fMAPK pathway O_LIRequired a properly folded SEA domain and conformational coupling to the cytosolic tail C_LIO_LIYapsin processing was required for SEA domain activation and turnover of the mucin cytosolic tail C_LI C_LI C_LIO_LIHOG pathway may sense protein misfolding as part of its activation mechanism. C_LIO_LISEA domains are conserved throughout fungal mucins and mammalian glycoprotein sensors suggesting a generalizable mechanism C_LIO_LIProtein deformation may provide information to survival pathways about environmental stress. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/723240v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1cd30f3org.highwire.dtl.DTLVardef@48c96corg.highwire.dtl.DTLVardef@9fffc2org.highwire.dtl.DTLVardef@504b1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Signaling pathways often share components yet activate different effector processes through mechanisms that remain unclear. The same mucin regulates two MAPK pathways (red and green), and the discovery of a conserved SEA domain provided insights into specificity mechanisms. In the fMAPK pathway that regulates filamentous growth, the mucin works in a classical manner, where an external signal (in this case underglycosylation by glucose limitation) transduces a signal to the cytosolic domain in cis. By comparison, the HOG pathway that responds to osmotic stress displayed a remarkable tolerance for mucin and SEA domain deformation. Protein variants that caused SEA domain misfolding, mislocalization, and degradation by ERAD retained function in the HOG pathway. Truncations that removed the cytosolic tail and transmembrane anchor were also functional. These phenotypes support a trans activation mechanism with external partners that was preferential for activation of the HOG pathway. SEA domain deformation may be induced by environmental stress as a trigger for the HOG pathway. Cells may detect misfolding of protein domains to gain information about environmental stress.