Cellular Microbiology

Vacuolar H+ ATPases (v-ATPases) are conserved proton pumps that support diverse biological functions through acidification of cellular organelles. The protozoan parasite Leishmania requires its v-ATPase for survival in the sand fly vector and mammalian host, but genetic mutants remain viable in vitro. To gain further insight into this conditionally lethal phenotype, we first mapped organellar localization of the v-ATPase by co-localisation imaging of fluorescently tagged v-ATPase subunits and organelle markers. The v-ATPase signal was strongest in the flagellar pocket region, consistent with enrichment in the contractile vacuole complex (CVC). To define the conditions that require a functional v-ATPase, deletion mutants were exposed to different stresses (pH, temperature, osmolality, dense culture). All tested deviations from standard culture conditions affected the mutants' growth rate, viability or both. Despite differences in phenotype severity, all stressors triggered the formation of a large autolysosome, positive for the autophagy marker protein ATG8 and the lysosomal enzyme cysteine peptidase A, indicating an arrest at the final step of autophagy. Measurements with the pH sensor pHLuorin2 showed that the luminal pH of the lysosomes was 5.6 in unperturbed promastigotes and 7.1 in v-ATPase mutants. These data support a canonical function for the Leishmania v-ATPase in lysosome acidification and autophagy, which is essential for parasite differentiation, and identify the poorly characterized Leishmania CVC as another major site of v-ATPase concentration.

Apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, reside within a specialized compartment known as the parasitophorous vacuole (PV) during their intracellular life cycle. The PV membrane (PVM), which derives from the host plasma membrane upon invasion, serves as a selective barrier that permits nutrient acquisition while shielding the parasite from host defense mechanisms. Although the protein composition of the PVM has been studied extensively, its lipid organization remains poorly understood. Using the quick-freeze, freeze-fracture replica labeling (QF-FRL) method, we quantitatively analyzed the transbilayer distribution of phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and GM3 ganglioside in the PVM of T. gondii and P. falciparum. Unlike host cell plasma membranes, where these lipids exhibit strict asymmetry--PtdSer and PtdEtn confined to the cytoplasmic leaflet and GM3 to the exoplasmic leaflet--we found that all three lipids were symmetrically distributed across both leaflets of the PVM. This striking loss of lipid asymmetry suggests that the PVM undergoes profound remodeling during infection. The presence of PtdSer and PtdEtn in the luminal leaflet may facilitate the binding of perforin-like proteins (PLP1s) during egress. These findings reveal a unique feature of the PVM that redefines our understanding of host-parasite membrane biology.

A cnidarian photosymbiont alga, Breviolum minutum, is an emerging model to study symbiosis due its ability to colonise host in absence of light, and amenability to genetic and physiological manipulations. This alga undergoes subcellular reorganisation in response to stress conditions such as elevated temperature and nutrient deprivation. However, subcellular visualisation of this alga is challenging because of its broad spectrum autofluorescence (400-700 nm) and relatively small size (6-8 m). We developed a super resolution imaging, Expansion Microscopy (ExM) workflow - a hydrogel-based technique for mechanical enlargement of cells, that reveals previously inaccessible subcellular features in B. minutum. This ExM workflow presents a set of thermic and enzymatic conditions which enables 4-fold expansion of B. minutum, optical clearing of autofluorescence as well as the removal of its thick cellulose rich cell wall. We implemented a recently described platinum (II)-based tri-functional linker 1, to retain in situ hybridised oligonucleotides targeted to 18S rRNA within ExM hydrogel and exploited its azide reactive group for post-ExM fluorophore labelling (DBCO modification). We observed actin patches (a cytoskeletal feature) and calmodulin (a calcium binding signalling protein) that are not previously visualised in B. minutum. This approach overcomes some of the long-standing problems in visualisation of B. minutum using commonly available reagents and commercially available low-cost ExM compatible chemistries. The broader uptake of this tool for the visualisation of diverse species of photosymbionts will pave the way for fundamental discoveries underpinning cellular reorganisation in formation and breakdown of symbiosis.

Photoendosymbiosis between the ciliate Paramecium tritobursaria and the green alga Chlorella variabilis provides a model for understanding stable photoendosymbiosis. A defining feature of this association is the perialgal vacuole (PV) membrane, a host-derived membrane that encloses each alga and prevents its digestion. However, the timing of PV membrane maturation remains poorly understood because of the lack of molecular markers to distinguish between immature and mature PV membranes. Previous studies have shown that the establishment of symbiosis proceeds through multiple regulated steps following algal uptake; however, the molecular maturation of the PV membrane has not been directly examined. Here, we report a monoclonal antibody that specifically recognizes the PV membrane in symbiotic P. tritobursaria. Time-course immunofluorescence analysis showed that the PV membrane antigen was absent in the early stages after algal uptake, appeared at 48 h, and was detected in all PV membranes by 72 h. The antigen persisted before and after synchronous PV swelling, an experimentally inducible state associated with the loss of normal PV membrane function, but was absent from the membranes surrounding the digested algae. Our findings provide the first molecular evidence that PV membrane maturation is a temporally regulated checkpoint during the establishment of photoendosymbiosis.

Fluorescence microscopy is central to the study of bacterial cell biology, multicellular behaviours, and host-pathogen interactions. Bright, robust and photostable labelling is required for bacterial identification, sorting and quantitative analysis, driving continuous development of state-of-the-art labelling tools. Here, we developed a multicolor fluorescent cell labelling toolkit for Gram-negative bacteria carrying the attTn7 site, using the opportunistic human pathogen Pseudomonas aeruginosa as a model. Cell labelling is achieved by constitutive chromosomal expression of genes encoding a choice of four novel fluorescent proteins, mNeonGreen, mJuniper, mLychee and mScarlet-I3, codon-optimised for P. aeruginosa. These reporters provide bright, stable fluorescence with minimal photobleaching and excellent spectral separation during long-term imaging of single cells, macrocolonies and biofilms. Chromosomal expression of mNeonGreen yielded brighter and more homogeneous labelling than expression of the same construct from a plasmid. Importantly, dual-color labelling of macrocolonies uncovered previously unrecognised phenomena of collective motility when two isogenic swarming populations interact. Finally, we demonstrate the applicability of our constructs in biologically relevant host-pathogen contexts by imaging both live and fixed P. aeruginosa-infected human airway epithelial cells. This versatile cell labelling platform enables reliable bacterial identification, segmentation, tracking, and quantitative fluorescence imaging across spatial and temporal scales, and is readily adaptable to most other Gram-negative bacteria as the attTn7 integration site is well conserved.

Host cell invasion by malaria parasites requires specific molecular interactions with host receptors. Plasmodium vivax merozoite invasion of reticulocytes is mediated by P. vivax Duffy binding protein (PvDBP) and its homolog, P. vivax erythrocyte binding protein (PvEBP). Here, we identify and characterize two novel P. vivax merozoite proteins, PvMP45 and PvMP36, which co- localize with PvDBP and PvEBP in the micronemes and bind reticulocyte receptors. PvMP45 and PvMP36 share high sequence identity with their P. knowlesi homologs, PkMP45 and PkMP36, which form a complex with other invasion related proteins. Field studies reveal that naturally acquired antibodies against PvMP36, PvEBP and PvDBP are associated with protection against clinical P. vivax malaria. We demonstrate that naturally acquired antibodies to PvEBP bind Fcy receptors and likely mediate protection by enabling opsonic phagocytosis. In addition, we show that combining antibodies against PvDBP and PvMP36 results in an additive invasion inhibitory effect against P. vivax blood stages. These results suggest that combining PvDBP, PvEBP and PvMP36 in a multivalent blood stage vaccine could elicit diverse immune mechanisms against P. vivax to achieve high efficacy. ImportanceAll the clinical symptoms of malaria are attributed to the blood stage of malaria parasites during which merozoites invade and multiply within red blood cells. A clear understanding of the host- parasite interactions that enable invasion can open paths for development of novel methods to block parasite growth and prevent malaria. Here, we identify and characterize two novel invasion related proteins from P. vivax merozoites that form an invasion complex and bind host RBC receptors. We demonstrate that antibodies targeting these proteins can block RBC invasion by P. vivax and naturally acquired antibodies that develop following P. vivax infection against one of these proteins are associated with protection against P. vivax malaria. These studies not only expand our understanding of the molecular mechanisms that enable host cell invasion by P. vivax but open new avenues for development of vaccines to protect against P. vivax malaria.

The erythrocyte sedimentation rate (ESR) is one of the most common and widely used laboratory diagnostic parameters in connection with inflammatory reactions and it is probable that every reader has already experienced a determination of their ESR. A rapid ESR is a non-specific parameter that provides information about the inflammatory process. Although the origins of this methodology date back to antiquity, the description of the process as the collapse of a percolating gel formed from erythrocytes has only recently been achieved. It was not yet known whether slow ESR has any medically relevant significance. Here we show a variety of clinical pictures that exhibit a systematically slow ESR (e.g., sickle cell disease, neuroacanthocytosis syndromes, chronic mountain sickness). Using a combination of measured data and physical modelling, we show how the accuracy and significance of ESR data can be increased. With this improved ESR (supraESR), we introduce a completely new, cost-effective diagnostic parameter, based on an established and easily automated measurement method, that enables low-cost screening for neuroacanthocytosis syndrome, a group of rare neurodegenerative diseases previously detectable only through complex diagnostic tests.

The morphology of cyanobacterial colonies plays a key role in harmful cyanobacterial blooms, with implications for their vertical migration, resistance against grazing, and light availability. In this study, we introduce the use of Optical Coherence Tomography (OCT) to investigate the three-dimensional morphology of cyanobacterial colonies. The technique enables non-invasive 3D imaging of colonies up to several millimeters in size, providing access to detailed mesoscale morphological features. Gas vesicles inside cells were shown to strongly improve image quality. We describe the sample preparation and image acquisition protocol, as well as an image processing pipeline that extracts mesoscale morphological features and provides a volumetric visualization of colonies. The method was tested for representative colonies of different cyanobacterial species while a dataset of volumetric images and measured mesoscale features was acquired for natural colonies of Microcystis. We demonstrate the utility of 3D imaging by quantifying the effects of irregular colony morphologies on their flotation velocity and the light availability within colonies. We anticipate OCT to become a key imaging technique to monitor populations of cyanobacterial colonies and investigate colony formation, with potential extensions to other colonial and aggregated organisms in freshwater and marine environments.

The sea anemone Aiptasia is a model system for understanding cnidarian loss of symbiotic algae under heat stress (bleaching). While Aiptasia polyps have been widely used to study this process, accurate symbiosis phenotyping grapples with discordant length scales: fine spatial resolution (~100 um) is needed across a whole organism (~5 mm). To address this, we consider small (~100 um), optically transparent Aiptasia larvae as a bleaching model suitable for whole-organism phenotyping by fluorescence microscopy with larvae classified as symbiotic when algae are localized within gastrodermal cells. To expedite phenotyping, we introduce a machine-learning (ML) image-analysis pipeline (SYMPHONY) designed for single-larva resolution analysis of intact larvae. SYMPHONY efficiently identifies the cellular location of internalized algae (accuracy: 79%, precision: 82%, recall: 79%, F1 score: 79%; training dataset composed of 1611 total objects). Additionally, SYMPHONY reports statistically significant larval bleaching under heat stress and corroborates manual phenotyping results, while significantly reducing operator labor from hours to minutes. The combination of the Aiptasia larvae model and the SYMPHONY pipeline aims to accelerate our understanding of symbiosis breakdown.

BackgroundA hallmark of the eukaryotic cell is the regulated transport between the nucleus and cytoplasm, which is mediated by a multi-subunit protein assembly called the nuclear pore complex (NPC). While its overall architecture has been preserved across eukaryotes, the NPC structure varies in different organisms, which appears to have tuned its function. Outside of a handful of model systems, the NPC has not been comprehensively studied. This is particularly true of species that are not closely related to well-studied models, such as apicomplexan parasites. Indeed, the evolutionary divergence of Apicomplexa has complicated facile prediction of NPC proteins in these organisms. Because of this, the NPC components remain largely unidentified, and therefore NPC cellular function in Apicomplexa is poorly understood. Principal FindingsHere we identified, experimentally validated, and functionally characterized protein components of the NPC in the apicomplexan parasite Toxoplasma gondii. By combining proximity biotinylation with careful bioinformatic analysis, we identified 16 previously uncharacterized proteins that localize to the Toxoplasma NPC. We demonstrated 8 of these proteins are essential to parasite replication. Importantly, we defined components of the mRNA export machinery, as well as Nups required for the stability and/or assembly of specific NPC subcomplexes. Consistent with the evolutionary distance between Toxoplasma and well-studied models, the majority of our newly validated NPC components show no clear homology to NPC proteins in yeast, animals, or plants. Moreover, we demonstrated that the Toxoplasma mRNA export machinery has a distinct composition from other well-established systems. Intriguingly, Sus1, a well-defined protein of the TREX-2 and SAGA complexes, is missing from the Toxoplasma genome. In contrast, others, such as Centrin-3, have been conserved in Toxoplasma, but are not required for mRNA export in the parasite. ConclusionOur work highlights the distinct composition of multiple subcomplexes of the Toxoplasma NPC and paves the way for future studies to provide high-resolution structural information on the parasites unusual NPC architecture.

The deformability of the red blood cell (RBC) is essential for microcirculatory flow and is profoundly altered in hemoglobinopathies and during infection with Plasmodium falciparum. While many mechanical tests have been developed to probe RBC-mechanics, the dynamics of cell shape recovery following large deformations remains poorly characterized. Here, we integrate microfluidic constriction assays, ultrafast imaging, and computer simulations to quantify time-resolved shape recovery of individual erythrocytes. We show that parasite infection is the primary determinant of RBC viscoelastic behavior. In wild-type (HbAA) erythrocytes, the relaxation time increases progressively from ring to trophozoite to schizont stages, consistent with parasite-induced membrane stiffening and enhanced membrane-cytoskeleton coupling. In contrast, sickle trait (HbAS) erythrocytes exhibit a distinct response: although deformation becomes increasingly irreversible during parasite maturation, the relaxation time after constriction remains largely unchanged. Analysis of a mutant parasite line with enlarged and sparsely distributed knobs revealed a significant increase in relaxation time, demonstrating that knob architecture modulates recovery kinetics. Together, these findings suggest that the coupling between membrane and cytoskeleton, which is strongly changed by the establishment of the knobs during an infection with Plasmodium falciparum, should have a strong detrimental effect on microcirculatory flow, which is however weakened by the sickle cell trait.

Germination percentage is an endpoint measure and therefore does not describe when an individual seed begins visible growth or how rapidly its radicle and plumule expand. We developed a time-resolved phenotyping workflow to quantify rice seed germination continuously in shallow-water culture. A single industrial camera moved along a 1 m rail and imaged three culture boxes at 1 h intervals for up to 80 h. The archive comprised 1,062 full-frame images and 6,372 seed-level repeated observations under the six-seed field-of-view configuration. A physical grid maintained seed identity through time and enabled individual regions of interest to be extracted. Whole-seed foregrounds were obtained with a pretrained U2-Net, and a masked RGB intensity rule separated newly emerging tissue from the darker hull. For each tracked seed, projected emerging-tissue area and interval growth rate were calculated. Three representative normally germinating seeds first showed measurable tissue at 48 h, yet subsequently followed distinct trajectories: final projected areas ranged from 2,605 to 4,700 pixels and peak interval growth rates ranged from 106.88 to 287.92 pixels h-1. B-1 accumulated 63.71% of its final visible area during 72-80 h, whereas B-3 accumulated 73.51% during 60-72 h. Thus, seeds with the same observed emergence interval can differ substantially in the timing and magnitude of post-emergence expansion. The workflow converts repeated images into biologically interpretable temporal phenotypes and provides a basis for nondestructive studies of rice seed vigor and germination heterogeneity.

Coxiella burnetii alternates morphologies to survive in two niches: the external environment and a degradative intracellular compartment. The small cell variant (SCV) is adapted for environmental persistence and transmission of Q fever to ruminants and humans. The large cell variant (LCV) is intracellular, and despite not being a major source of transmission, is infectious in vitro. When modeling infection, researchers typically apply a mixed population of these cell types as inocula. As this practice does not mimic natural infection, it may confound our understanding of early Q fever infection events. We separated SCV and LCV by density gradient centrifugation and compared their replication in primary murine macrophages and a fibroblast cell line. SCV inocula replicated more efficiently than LCVs in both host cell types. LCV replication was delayed for four days in macrophages compared with SCV inocula, which had completed logarithmic growth by that time point. We found no difference in pathogenic vacuole size, but there was a modest difference in their respective bacterial burdens. Interestingly, IL-6 and CXCL2 secretion was significantly elevated in LCV-infected macrophages as compared to SCV at 24 hours, suggesting a difference in the host response to each. This is the first study to demonstrate that C. burnetii developmental status influences the progression of infection.

Otolith organs are specialized structures of the vertebrate inner ear that provide the inertial mass required for maintaining equilibrium. Mammals possess two otolithic organs, the utricle and the saccule, whereas birds and other non-mammalian vertebrates also retain a third one, the lagena, whose development remains poorly understood and whose function is still debated. In birds, the lagena contains thousands of calcium carbonate biomineral particles, termed otoconia. Here, we reconstruct the developmental crystallization of lagena otoconia in the Japanese quail (Coturnix japonica) throughout embryogenesis. We combine multiscale imaging with structural, compositional, and crystallographic analyses across length scales. We show that lagena mineralization precedes cranial bone formation and proceeds predominantly through the growth of existing otoconia rather than continued nucleation. Otoconia develop through progressive particle growth, alignment, and fusion within a pre-existing organic compartment while maintaining a persistent central core, ultimately forming magnesium calcite biominerals. This maturation is accompanied by progressive nanoscale densification, transforming early mineral deposits into mature hierarchical crystals. This work establishes a developmental model of avian otoconia formation and provides new insights into how hierarchical calcium carbonate crystals are assembled during vertebrate development. Statement of significanceOtoliths are the only calcite based biomineral in our body that has a physiological function, yet their developmental assembly remain incompletely understood. While the utricle and saccule have been extensively investigated across vertebrates, the lagena, a third otolithic organ lost during mammalian evolution, has received comparatively little attention. Here, we combine multiscale imaging and materials characterization to reconstruct the developmental crystallization of lagena otoconia in the Japanese quail. We establish how hierarchical magnesium calcite biominerals emerge through coordinated mineral growth, structural maturation, and crystallization, providing a developmental framework for avian otoconia formation and new insights into the assembly of vertebrate calcium carbonate crystals.

Background Visceral leishmaniasis (VL), caused by Leishmania donovani (LD), is increasingly associated with the insect-restricted trypanosomatid Leptomonas seymouri (LS), which harbours the RNA virus Leptomonas seymouri narna-like virus 1 (Lepsey NLV1). Our recent study demonstrated that LS co-infection with LD enhances survival of murine (RAW 264.7) and mammalian (THP-1) macrophages and augments LD and LS persistence compared to LD or LS mono-infection in vitro. However, the in vivo fate of LS and its viral endosymbiont during chronic VL remains poorly understood. This study investigated the long-term dynamics of parasite persistence, tissue dissemination and viral maintenance during experimental mono- and co-infection. Methods and Findings BALB/c mice were infected with LD, Lepsey NLV1-positive LS, virus-positive AG83 isolate, or LD: LS co-infections (2:1, 5:1 and 10:1) and monitored for up to seven months. Parasite burden, species composition and viral load were quantified using ITS1 qPCR, densitometry, nested RT-PCR and qRT-PCR, supported by microscopy and immunofluorescence assay. LS established productive visceral infection independently, with parasite burdens exceeding the infecting inoculum, indicating active in vivo replication. Co-infection, particularly at a 10:1 LD: LS ratio, promoted the greatest long-term parasite persistence in visceral organs. Temporal analysis revealed early predominance of LS followed by progressive recovery of LD during chronic infection. Lepsey NLV1 was detected in visceral organs and blood for at least up to five months. Morphological analyses demonstrated intracellular LS amastigote-like forms in murine macrophages and transformation of splenic parasites into promastigotes, confirming parasite viability within mammalian tissues. Conclusions These findings demonstrate sustained visceral persistence of Lepsey NLV1-positive LS in mice and identify dynamic host-parasite-virus interactions that reshape infection during chronic co-infection. This work challenges the conventional view of VL as a strictly mono-parasitic disease and highlights a previously underappreciated tripartite interaction with potential implications of LS and its virus endosymbiont for VL pathogenesis.

The metacestode of Echinococcus multilocularis is the proliferative larval stage responsible for alveolar echinococcosis and displays remarkable capacities for long-term growth, regeneration and development within the host. Despite its medical relevance, the cellular composition and molecular organization of this stage remain incompletely characterized. Here, we generated the first single-cell transcriptomic atlas of the E. multilocularis metacestode, resolving 26 transcriptionally distinct cell populations. The atlas recovered the major cell types previously described in the germinal layer, including germinative, tegumental, muscle, neuronal and putative storage cells, and revealed substantial molecular heterogeneity within several of these compartments. In particular, germinative cells segregated into distinct transcriptional states, ranging from a population enriched in markers associated with an undifferentiated germinative state to populations displaying early tegumental- or muscle-associated transcriptional programs. Notably, one of these states was strongly enriched in an isolate retaining the capacity for brood capsule and protoscolex formation but was nearly absent from a developmentally deficient isolate, suggesting a possible association between germinative-cell heterogeneity and developmental competence. Differentiated populations likewise displayed distinct molecular specializations, including developmental signaling and extracellular-matrix programs in muscle cells, microtubule-associated and transporter expression in tegumental populations, and metabolic specialization in putative storage cells. Spatial validation by whole-mount in situ hybridization, EdU labeling and immunofluorescence established molecular markers for major cell populations and revealed stage-specific expression patterns between metacestodes and protoscoleces. Together, these data uncover an unexpected level of molecular and cellular heterogeneity within the morphologically simple metacestode germinal layer and establish a cell-resolved framework for investigating stem-cell organization, differentiation and developmental plasticity in this medically important parasite.

Malaria infections are initiated by mosquito bites, during which Plasmodium sporozoites are injected into the host skin. Sporozoites migrate rapidly to find and enter blood capillaries and ultimately invade hepatocytes. Sporozoite migration and invasion is mediated by the transmembrane protein thrombospondin-related anonymous protein (TRAP), which links the extracellular substrate to the actomyosin complex powering gliding motility, while the abundant, GPI-anchored circumsporozoite protein (CSP) covers most of the parasite membrane and modulates adhesion. Both proteins are secreted onto the parasite surface and deposited in a membranous trail originating at the parasite rear. The surface dynamics of these essential sporozoite proteins and the mechanism of deposition, however, are not understood. Here, using orbital total internal reflection fluorescence microscopy (TIRF), we reveal the dynamics of TRAP adhesion site formation and disassembly as well as CSP and TRAP deposition rates. We find that TRAP assembles into distinct adhesion sites, which then undergo retrograde translocation as the sporozoite moves forward. Around half of the TRAP adhesins, together with CSP, remain associated in small membrane droplets on the substrate after the sporozoite has disengaged from the adhesion site. These droplets seem to originate from nanotubes, that presumably decay under high tension. Strikingly, we observe a change in actin filament accumulation if proteolytic cleavage of TRAP is inhibited, providing the first visual evidence for outside-in signaling in sporozoites. Our study reveals a relation between adhesion dynamics and trail formation in Plasmodium sporozoites that might also be relevant for other cell types.

Functional type III secretion in Gram negative bacteria relies on precise substrate targeting and a strict order of secretion with early, intermediate, and late substrates. Type III secretion chaperones facilitate these processes by maintaining substrates in a partially unfolded, secretion-competent state and serving as order-specific targeting factors. Early needle filament assembling substrates are chaperoned by none or class III chaperones, intermediate translocator-type substrates by class II and late effector-type substrates by class I chaperones. In case of hydrophobic transmembrane effectors, chaperones may also serve to prevent erroneous mistargeting of these substrates to the bacterial inner membrane. Here, we characterized the Salmonella transmembrane effectors SseF and SseG and their chaperone SscB, encoded in the operon sscB-sseF-sseG, in order to gain a deeper understanding of the underlying molecular requirements of targeting of this special class of substrates. We show that the gene linkage of SscB and SseF is critical for these proteins stability and SseF secretion. Counterintuitively, SscB revealed to feature a class II chaperone structure with a class I chaperone function. Likewise, SseF and SseG harbour conserved, translocator-like chaperone-binding motifs (PXI/LXXP) but were secreted as late substrates, independent of the gatekeeper protein SsaL. These findings challenge the current chaperone classification and our understanding of the molecular basis of the hierarchy of substrate secretion. They show that chaperone structure is not a sufficient molecular determinant for the correct order of substrate secretion.

Polarization vision subserves diverse biological functions, such as navigation, communication and target-motion detection. Regarding the detection of biological targets, studies on semi-terrestrial crabs suggest that polarization and intensity contrast are processed in separate visual channels. Information about the polarization contrast of targets would be extracted independently of intensity contrast, and the two signals combined downstream in the visual system. However, understanding how the information about these visual attributes is processed and integrated has been limited, as it is technically challenging to present visual stimuli in which both the polarization and the intensity contrast of a stimulus are controlled. Here we developed a monitor screen that allows us to present stimuli in which both contrasts can be controlled. Thus, to study how polarization and intensity information is processed to increase target detection, we presented moving stimuli with controlled polarization and intensity contrast while recording the cardiac response of the semi-terrestrial crab Neohelice granulata as a sensitive readout of its visual perception. Our results suggest that Neohelice possesses similar sensitivity to vertically and horizontally polarized light; thus, previously reported responses of the animals to polarized stimuli in which figure and background have the same intensity are likely accounted for by the comparison of two polarization channels. In addition, we determined that a moving polarization-only stimulus has a salience equivalent to that of an intensity-only stimulus with a Michelson contrast of 0.51. Finally, we studied how polarization and intensity contrast information is integrated, and found that polarization contrast increases the salience of an intensity-contrast-based target mostly when its intensity contrast is low, i.e. when information about intensity contrast is more ambiguous.

The phylum Chlamydiota comprises obligate intracellular bacteria characterized by a highly conserved, biphasic developmental cycle. This cycle involves the transition between the infectious, metabolically quiescent elementary body (EB) and the non-infectious, replicative reticulate body (RB). While the morphological transitions of the developmental cycle are well-documented, the regulatory mechanisms governing these phenotypic shifts remain poorly understood. A primary candidate for this regulation is Euo, a conserved, phylum-specific helix-loop-helix transcription factor hypothesized to repress late-cycle genes and prevent premature differentiation. In this study, we employed CRISPR interference (CRISPRi) to knockdown euo expression in Chlamydia trachomatis to further elucidate its role in developmental regulation. Unexpectedly, euo knockdown did not significantly disrupt the primary developmental cycle; progression through RB replication, the formation of intermediate bodies (IBs), and the kinetics of late-gene expression remained largely comparable to wild-type. However, we observed a significant reduction in the production of infectious progeny. Detailed analysis revealed that while EBs were still produced and capable of entering host cells after knock down of euo, these EBs exhibited dysregulated gene expression during the germination phase of a new infection cycle. Consequently, these bacteria failed to establish a productive secondary infection. These results suggest that rather than acting as a developmental switch for differentiation during the initial infection, Euo is essential for the proper programming of EBs, ensuring transcriptional competence upon re-infection of a host cell.