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Screening for, detecting, and managing pregnancy hypertension is a core function of antenatal care. To reduce both training requirements and the risks of measurement error in blood pressure (BP) values, automated and semiautomated BP devices have been validated in pregnant women with normal BP and pregnant women with hypertension and introduced for serial antenatal measurement of BP. The study aimed to (1) determine whether or not repeated BP measurements reduced the presence of terminal digit preference and (2) discern whether or not there was evidence of threshold avoidance in the Community-Level Interventions for Preeclampsia (CLIP) trials compared with the purely observational Pregnancy Care Integrating Translational Science, Everywhere (PRECISE) cohorts. The BP 3AS1-2 and CRADLE Vital Signs Alert low-cost Microlife BP devices were used by trained research staff in the CLIP trials conducted in India, Mozambique, Nigeria (pilot trial only), and Pakistan and the PRECISE cohorts of unselected pregnant women and nonpregnant women of reproductive age recruited in the Gambia, Kenya, and Mozambique. Both devices algorithmically calculate systolic blood pressure and diastolic blood pressure values displayed on digital read-outs. All BP readings were entered manually into a digital platform, which averaged them as the BP for that visit; the first and second readings were averaged unless they were more than 10 mm Hg different, which triggered a third reading, and the second and third readings were averaged. A total of 51,875 participants had their BP measured 438,404 times. Using raw BP values, there was terminal digit preference (129,539/911,500, 14.21% vs 10%; P<.001 values ended in zero). A total of 28,929 out of 437,446 (6.61%) dBP values were 62 mm Hg, compared with 9310 of 195,349 (4.77%) from the averaged values (P<.001); errors were obviated by averaging BP values. There was evidence of both threshold preference and avoidance in the CLIP trials and the PRECISE cohort. Given the excess of 62 mm Hg values, there is a shared inherent algorithmic error in the calculation of dBP in the BP 3AS1-2 and CRADLE Vital Signs Alert devices. Averaged BP measurements are important to reduce the impact of user errors in manually recording BP values. We recommend that automated and semiautomated BP devices should be connected wirelessly to automatically transfer readings to digital health records to further optimize care.

Chlamydia trachomatis is a major human pathogen responsible for the most prevalent bacterial sexually transmitted infections. Given its high prevalence, potential for serious complications and high economic burden, understanding the pathogenesis of C. trachomatis infections is crucial for developing effective prevention and treatment strategies. Mucosal epithelial cells serve as the primary replicative niche and play a central role in infection. Historically, research has relied heavily on cancer-derived cell line models to study the interactions of&#xa0;C. trachomatis with host cells. Although these models have provided important insights, they fail to fully recapitulate key aspects of human physiology, including apical-basal polarity, a fundamental feature that shapes epithelial functions. Limited studies using polarized in vitro epithelial systems have uncovered unique mechanisms by which C. trachomatis interacts with host cells. In this review, we summarize the current, albeit limited, knowledge on the role of epithelial polarity during C. trachomatis infection and highlight this understudied aspect of chlamydial biology.

Type IV secretion systems (T4SS) are found in both monoderm and diderm bacteria. The broad-host-range conjugative plasmid pIP501 from Enterococcus faecalis harbors a T4SS encoding 15 tra genes responsible for the spread of antimicrobial resistance genes among diverse G+ pathogens. Eight Tra proteins (TraB, TraCB3, TraF, TraHB8, TraI, TraK, TraLB6, and TraMB8) are postulated to form the mating pair formation (MPF) complex representing the central DNA translocation pore. One of these proteins is TraF, a 52.8&#xa0;kDa transmembrane protein, which lacks any homologs in other well described T4SSs. In this study, TraF was proven to be an essential conjugative transfer protein. The TraF pulldown co-eluted all Tra proteins except TraGB1 and TraN. Bacterial-two-hybrid assay showed a strong interaction between TraF and TraMB8. We present a 1.25&#xa0;&#xc5; resolution crystal structure of the N-terminal domain of TraF, which adopts a pseudokinase fold. AlphaFold predictions of full-length TraF with membrane mimetics show a transmembrane protein with two distinct soluble domains. FoldSeek revealed a strong similarity to YukC (EssB), a transmembrane pseudokinase from type VII secretion system (T7SS). YukC was shown to function as an interaction hub by mediating contacts between its pseudokinase domain and other T7SS proteins as part of the central membrane core complex. We postulate that TraF might play an important role in T4SS complex formation.

Bacteroidota, a diverse phylum of bacteria, includes classes whose members are increasingly recognized for their significant contributions to host health, particularly through their antimicrobial properties. This study investigates the functional diversity of 42 new Bacteroidia and Sphingobacteriia strains enriched and identified from diverse hosts, including mouse ceca and human stool samples. Using 16S rRNA gene sequencing, we phylogenetically characterized the strains of the genera Bacteroides, Phocaeicola, and Sphingobacterium and assessed their functional properties related to potential beneficial functions. The strains were evaluated concerning their ability to inhibit biofilm formation of the World Health Organization-declared clinically significant pathogens, including Gram-positive Staphylococcus aureus and Staphylococcus epidermidis, Gram-negative Klebsiella oxytoca and Pseudomonas aeruginosa, and the eukaryotic yeast Candida albicans. Additionally, we investigated bile salt hydrolase and quorum-quenching (QQ) activities of the strains, as these functions contribute to microbial community interactions and host-microbe dynamics. Our findings demonstrate that all examined Bacteroidota strains consistently exhibit a capacity to inhibit biofilm formation but to different extents. Furthermore, 14 strains showed QQ activity, and 39 bile salt hydrolase activity, indicating functional diversity among the isolates. High biofilm inhibition as well as QQ activity against both autoinducers, AHL and AI-2, were predominantly observed in Bacteroides caecimuris and Bacteroides muris. These traits suggest that such strains may play important roles in shaping microbial communities and interfering with pathogens and their communication. Overall, this study provides valuable insights into strain-specific functions that could support future microbiome-based strategies for pathogen control and host health modulation.

[This corrects the article DOI: 10.1093/femsml/uqaf041.].

Guanosine tetra- and pentaphosphate ((p)ppGpp) are one of the key players in the stress response of bacteria. Accumulation of these alarmones activates the stringent response, usually triggered by different nutritional stresses. For Pseudomonas putida, there is only limited data available on the importance of the stringent response in stress situations. Also, in recent years, different specific phage defence systems have received much attention, but little is known about the involvement of stringent response in phage infection. Here, we show that P. putida PaW85 (p)ppGpp0 is prototrophic and tolerates chemical stress well. However, in the stationary phase P. putida cells deprived of (p)ppGpp have impaired membrane integrity. In addition, we conducted a large-scale screening of stringent response effects on phage infections using the CEPEST phage collection. We tested 67 phages of 22 different species and revealed that the lack of (p)ppGpp has opposing effects on phage infection with nearly half of the tested phages showing higher infection efficiency on the (p)ppGpp0 cells, whereas the other half shows reduced infection. We show that the differences in phage infection efficiency for phages Aura and Amme-3 are not caused by adsorption rate differences, but alterations in downstream steps of the infection cycle-prolonged latent period in the absence of (p)ppGpp or unproductive infection in the presence of (p)ppGpp. Altogether, results indicate that the role of stringent response in phage infection is highly diverse, and over half of the times the presence of (p)ppGpp facilitates phage infections rather than protects the cells.

In proteome studies, the application of alternative proteases, exclusively or in addition to trypsin, often increases protein sequence or proteome coverage. It has recently been shown that, in particular, the analysis of small proteins benefits from such multi-protease approaches. However, selecting the most optimal combination of proteases either requires laboursome experiments or the decision of an experienced user, which might be biased. In this manuscript, we present a protease score that enables the objective comparison of multiple-protease digestions and a Python-based tool named CoMPaseD (Comparison of Multiple Protease &#xa0;Digestions), which utilizes Monte-Carlo simulations to predict this score for a user-defined set of proteases and any combination of these. By analysis of the small proteomes of the two model organisms Bacillus subtilis and Methanosarcina mazei with five proteases and different experimental setups, we demonstrate a good correlation between experimentally derived and predicted scores. This highlights the broad applicability of CoMPaseD, which can effectively guide the selection of proteases to enhance the characterization of specific subsets of the proteome, e.g. based on factors such as protein size, localization or isoelectric point. CoMPaseD is freely available at https://github.com/MicrobialProteomics/CoMPaseD.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#x3ba;B) represents a family of important transcription factors in innate immunity. We have previously reported that the gastric pathogen Helicobacter pylori needs the actin-binding protein cortactin for efficient interleukin-8 (IL-8) secretion, which requires NF-&#x3ba;B activation. However, it remained unknown, which exact cortactin signaling mechanism contributes to IL-8 release. In fact, H. pylori profoundly activates NF-&#x3ba;B in wild-type AGS gastric epithelial cells by the effector molecule adenosine diphosphate (ADP)-&#x3b2;-d-manno-heptose (ADPH) in a type IV secretion system-dependent manner. However, the injected CagA protein might contribute to NF-&#x3ba;B activation. The ADPH-stimulated canonical NF-&#x3ba;B cascade involves alpha-kinase 1 and adapter protein TRAF-interacting protein with forkhead-associated domain (TIFA) to activate inhibitor of kappa B (I&#x3ba;B) kinases (IKKs), followed by phosphorylation-dependent degradation of I&#x3ba;B&#x3b1; and subsequent nuclear translocation of p65 NF-&#x3ba;B and IL-8 release. Here, we show that infection of cortactin knockout cells leads to reduced activation of focal adhesion kinase (FAK) and c-Sarcoma (Src) kinase resulting in diminished phosphorylation of IKK&#x3b2;&#xa0;at tyrosine residue 199 and subsequently phosphorylation of p65 at serine residue 536, both of which are associated with downregulated NF-&#x3ba;B activity. Our results were further supported using FAK and TIFA knockout cells and treatments with purified ADPH and overexpression of CagA, showing cumulative effects in wild-type, but not in knockout cells. These data demonstrate that ADPH-dependent NF-&#x3ba;B activation and IL-8 secretion are enhanced by CagA. Together, we present here a novel CagA>cortactin>FAK>Src>IKK&#x3b2;&#xa0;signaling cascade, contributing to proinflammatory responses by H. pylori.

Quantitative information on protein abundance is crucial to understand biological processes and is therefore frequently gathered in proteomic studies. However, the quality of a quantitative proteomic dataset is greatly affected by the number of missing values, which need to be minimized to produce robust and meaningful data. In this context, small proteins (&#x2264;100 amino acids) pose specific analytical challenges, which hinder their efficient identification and quantitative characterization in complex proteomes. In this study, methods for sample preparation and MS-data processing are systematically evaluated for their contribution to identification and quantification of small proteins of Clostridioides difficile 630 &#x394;erm. Results show that small protein enrichment can enhance the number of identified and quantified proteins also for low abundant small proteins. Through application of spectral libraries for identification of MS spectra the number of robustly quantified proteins is increased and a lower limit of their detection is reached. Additionally, the dataset presented here is currently the most comprehensive protein repository for C. difficile covering 84.7% of the predicted proteome and 61.4% of all predicted small proteins of this important pathogen.

Salmonella enterica is a facultative intracellular pathogen capable of surviving within host cells, where it faces a sophisticated immune arsenal. Within the phagosomal compartment, the bacterium encounters significant stress from copper and reactive chlorine species like N-chlorotaurine (N-ChT) generated during the oxidative burst. We investigated the regulatory mechanisms enabling Salmonella to adapt to this dual copper/oxidative stress, specifically focusing on the regulation and function of CueP. This periplasmic protein was previously proposed to bind copper ions and to transfer them to the superoxide dismutase SodCII. Here, we demonstrated that copper specifically triggered the CueR pathway and that N-ChT activated the Cpx pathway; simultaneous exposure to both stresses resulted in maximum cueP expression levels. Moreover, CueP was shown to be important for copper resistance in the absence of the multicopper oxidase CueO and exhibits high thermostability in the presence of copper. Additionally, in a &#x394;cueO background, copper is sufficient to activate the Cpx pathway, ensuring robust cueP induction even without external oxidative signals. These findings establish a direct molecular link between a host antimicrobial agent (N-ChT) and the activation of the Cpx-CueP axis, revealing a new layer of bacterial adaptation to innate immunity. Moreover, they highlight an integrated response strategy contributing to bacterial adaptation to the dual copper/oxidative stress.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated protein (Cas) systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR-Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56&#x2009;343 CRISPR arrays across 25&#x2009;628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting higher spacer conservation. This observation corroborates recent findings indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations might play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.

Natural killer (NK) cells contribute to the innate immune system and are pivotal for the defence against opportunistic pathogens, including fungi. Aspergillus fumigatus (AF), a filamentous mold, can cause invasive pulmonary aspergillosis in immunocompromised patients, e.g. in patients after allogeneic stem cell transplantation (alloSCT). In this pilot study, we challenged NK cell samples from alloSCT recipients collected 90, 120, and 180 days after transplantation and from healthy individuals with AF and characterize the proteome response differences. We identified 2259 differentially abundant proteins between the NK cell proteomes of alloSCT recipients and healthy individuals. Among these, 1118 proteins were differentially abundant at all time points and 1931 proteins specifically at day 180 post-alloSCT. Following stimulation of NK cells with AF, we found a profoundly different early proteome (day 90,&#xa0;n=1652 proteins), while at day 180, only 77 proteins remained significantly differentially abundant. We identified, among others, a major differentially abundant protein cluster related to IL27RA (including OAS, STAT1, and MX). Furthermore, for selected markers [granzyme A (GZMA), Neural Cell Adhesion Molecule 1 (NCAM1/CD56), perforin-1 (PRF1)], we confirmed our proteome data by flow cytometry in NK cells from an independent second patient and healthy individual cohort. In conclusion, we demonstrate the advantage of combining comprehensive proteomic profiling with targeted flow cytometry to investigate NK cell responses to AF. Our data analysis connects STAT1 with IL27RA as well as granzyme, IFNg, and NCAM1 activity, which may be exploited towards future therapeutics warranting confirmation in larger study cohorts.

In heterogeneous environments, the hyphae of filamentous fungi and oomycetes can facilitate the dispersal of other microorganisms. The use of these "fungal highways" (FH) is regulated by both physical and biological factors with their interplay resulting in variable capabilities of different microbes to establish FH. Several devices have been developed to test the movement of bacteria across mycelium. However, these methods are usually time-consuming and cannot be applied at a large scale. In this study, we developed 3D-printed experimental devices that physically separate two environments while allowing hyphal networks to act as bridges for bacterial movement. The final design allows for the simultaneous testing of up to 10 pairs and the inclusion of any culturing media. With these devices, we investigated how fungal-bacterial pairing, nutrient conditions, and inoculation strategies influence FH formation. Bacterial transport was limited in nutrient-rich media but increased under poorer nutrient conditions, consistent with enhanced exploratory growth of the mycelium. Both cis- and trans-inoculation supported FH formation, although bacterial arrival was delayed in the absence of co-inoculation. The devices were used to demonstrate that transport of bacteria by FH was relevant for the colonization of a natural substrate. Finally, we established a novel in planta assay to evaluate FH formation during host colonization. This assay demonstrated that Fusarium graminearum can transport bacteria during wheat spike colonization. Together, these results provide accessible, scalable tools to study hyphal-mediated bacterial dispersal and highlight the combined role of biological specificity and nutrient context in the establishment of FH.

The establishment of a replicative niche in the hostile environment of the host presents an enormous challenge for pathogens. Intracellular pathogens such as Brucella spp., the Gram-negative causative agents of Brucellosis, must subvert diverse host functions to ensure survival and replication. One of the key adaptations to achieve this is the translocation of effector proteins into host cells via its type 4 secretion system (T4SS). But effector identification in Brucella is particularly challenging, as previously identified effectors lack a conserved translocation signal, exhibit variable requirements for translocation, and in some cases appear to be translocated in a T4SS-independent manner. Here, we demonstrate that a subset of previously described T4SS effector proteins associates with outer membrane vesicles (OMVs) in different Brucella species. Most of these effector proteins encode predicted signal peptides for periplasmic export or transmembrane domains. Among them, BspC and VceA carry functional signal peptides that direct their export into the periplasm in a Sec-dependent manner. From the periplasm, these proteins are subsequently secreted into the extracellular milieu, likely via the formation and release of OMVs. Our findings provide new insights into protein secretion by Brucella, suggesting that OMVs may represent an alternative secretion pathway to the T4SS.

Intracellular Gram-negative pathogens employ either type IVA or type IVB secretion systems (T4SSs) to translocate effector proteins into host cells, where they modulate cellular processes to facilitate infection and promote intracellular survival. Roughly one-third of these effectors harbor hydrophobic transmembrane domains and are thus destined for integration into host cell membranes during infection. Many of these transmembrane domain-containing effectors (TMEs) localize to the membrane of the pathogen-containing vacuole, thereby contributing to its formation and remodeling. Despite the biological relevance of TMEs, the detailed molecular mechanisms governing their translocation via T4SSs and subsequent membrane integration in the host cell remain insufficiently understood. In this review, the biophysical characteristics of T4SS-secreted TMEs are systematically examined, including predictions of membrane topology and hydrophobicity. These analyses are then contextualized through comparison with recent structural analysis of both T4ASS and T4BSS machineries, as well as with mechanistic principles of eukaryotic membrane protein biogenesis. This integrative approach enables the conceptual reconstruction of the potential pathways by which TMEs are translocated through the T4SS and subsequently targeted and inserted into host membranes, offering new mechanistic insights into the poorly understood handling of bacterial TMEs from both the pathogen and host perspectives.

Understanding host-pathogen interactions at the molecular level requires methods capable of linking spatial context with proteomic information. Here, we present an integrated workflow combining matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and laser microdissection (LMD)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate Aspergillus fumigatus infection in murine lung tissue. Consecutive formalin-fixed, paraffin-embedded (FFPE) tissue sections were used for spatially resolved MALDI-MSI and subsequent LC-MS/MS analysis of laser-microdissected fungal-infected and non-infected regions. MALDI-MSI revealed reproducible m/z features specifically associated with infected areas. Corresponding tissue microregions were microdissected and analyzed by proteomics to identify candidate proteins underlying these spatial signals. Comparative proteomics of fungal-infected with non-infected alveolar lung regions via LC-MS/MS identified host proteins involved in leukocyte recruitment, inflammatory signaling, and reactive oxygen species formation, including a 424-fold increase in formyl peptide receptor 2 (Fpr2) during fungal invasion of the lungs. Fungal regions were also enriched in proteins encoded by the gliotoxin biosynthetic gene cluster. Spatial and proteomic data were linked by matching theoretical peptide-adduct masses to MALDI-MSI features, using a semi-quantitative scoring system to prioritize protein assignments. Fungal regions showed contributions from both host and pathogen proteins. This workflow establishes a conceptual basis for spatial proteomics of host-pathogen-interactions in fungal infections and enables association of characteristic m/z signals with plausible protein candidates.

Pyrobaculum arsenaticum and P. aerophilum are two hyperthermophiles that belong to the phylum Thermoproteota (also known as Crenarchaeota), order Thermoproteales. Pyrobaculum arsenaticum is an obligate anaerobe, whereas P. aerophilum is a facultatively aerobic organism. Both species have been described as capable of autotrophic growth with molecular hydrogen. Because their genomes lack genes encoding key enzymes for known autotrophic CO2 fixation pathways, they have been discussed as organisms that may use unknown pathways. To establish reliable autotrophic cultures, we gradually reduced the supplied concentrations of yeast extract but, in our hands, autotrophy was not attainable for either of the two species. Analysis of the 13C-labelling of the biomass of the obtained mixotrophic cultures of P. arsenaticum grown on 13CO2 + H2 (20:80, v/v), using isotopologue profiling, revealed that their amino acids contained <30% of 13C. Amino acids were mainly labelled only in carboxyl groups, demonstrating their purely heterotrophic nature. Our data suggest that the ability to grow autotrophically in currently known Thermoproteales is strictly correlated with the presence of the genes for the dicarboxylate/4-hydroxybutyrate cycle. We discuss the reasons that may lead to misinterpretation of the data on the ability of prokaryotes to grow autotrophically.