Abstract Bacterial growth patterns are generally considered to be constrained and species-specific. Here, we show that Sphingobium yanoikuyae exhibits exceptional plasticity in envelope architecture and morphogenesis driven by a glycosphingolipid (GSL)-based outer membrane and an atypical peptidoglycan structure. At 30°C, cells expand asymmetrically via a rare bipolar envelope synthesis mode, whereas growth at 37°C triggers a transition toward longitudinal elongation accompanied by increased outer membrane vesiculation, indicating a reversible reprogramming of morphogenesis under host-like conditions. This switch is supported by rapid outer membrane dynamics, including high membrane fluidity and fast redistribution of envelope components. Despite these pronounced morphological changes, peptidoglycan composition remains conserved and unusually short, suggesting that growth plasticity is governed primarily by spatial regulation rather than changes in cell wall chemistry. Genetic analyses further identify essential roles for proteins involved in outer membrane–peptidoglycan and outer membrane–inner membrane coupling, as well as GSL transport systems, and core cell wall synthesis machinery, while revealing extensive redundancy in envelope remodeling enzymes. Together, these results establish S. yanoikuyae as a model for extreme envelope adaptability, where a highly fluid outer membrane and structurally unconventional peptidoglycan enable reversible transitions between distinct growth programs, potentially shaping environmental fitness and host-associated interactions.

Despite decades of investigation into bacterial pathogens, the conditions met by intracellular bacteria are still unclear. These conditions can include access to nutrients, such as amino acids, and exposure to toxic compounds, like copper. To investigate the ability of Brucella abortus, a facultative intracellular pathogen responsible for a major zoonosis, to cope with copper, we performed a Tn-seq analysis to identify copper-sensitive mutants. Unexpectedly, we realized that classical copper resistance systems (involving CopA and CueO homologs) do not appear to be robustly needed, while histidine and purine biosynthesis pathways are crucial to cope with copper. We show that hisA, hisB, hisC, and hisD mutants are auxotrophic for histidine and sensitive to copper. This suggests that the reported attenuation of his mutants in macrophages could be based on auxotrophy and/or copper sensitivity. Therefore, we generated suppressor strains with a restored resistance to copper for hisC, but still auxotrophs for histidine. Our data suggest that this suppression is due to the overproduction of a homolog of OppA, a periplasmic oligopeptide-binding protein. Analysis of these suppressors shows that the absence of histidine biosynthesis capacity, and not copper sensitivity, is required for optimal growth of B. abortus in macrophages.IMPORTANCEInvestigating conditions in which intracellular bacteria grow inside host cells is challenging and often involves the characterization of attenuated bacterial mutants obtained by screening. But a single mutant can display two different phenotypes related to intracellular conditions. It was the case for histidine auxotrophs of Brucella abortus, an important zoonotic pathogen. These histidine auxotrophs are attenuated in a macrophage cell line, and they are also sensitive to copper stress. Using a suppressor strain still auxotroph for histidine but with an improved resistance to copper, we show that histidine auxotrophy, and not sensitivity to copper excess, is the main cause of attenuation in the conditions tested here.

BackgroundAcinetobacter baumannii is an opportunistic bacterium that causes serious nosocomial infections, including pneumonia and bacteremia, especially in immunocompromised individuals.MethodsHere, we first evaluated the protective efficacy of a vaccination protocol with the heat-killed (HK) A. baumannii strain LAC-4 in various models of immunodeficient C57BL/6 mice challenged with pulmonary infection by LAC-4. We then examined the ability of HK LAC-4 to stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors in vitro.ResultsWe observed that mice deficient in Th1 (IL-12p35-/-, IFN-γ-/-), Th17 (IL-17RA-/-) and T cells (δTCR-/-, TAP1-/-, CD3-/-) display higher susceptibility to LAC-4 infection, but that our protocol improves their resistance. In contrast, vaccinated B cell-deficient (MuMT-/-) mice appear unable to control the infection, demonstrating that humoral immunity is essential to vaccine protection. Vaccination of wild-type mice with an HK ΔitrA strain deficient for capsule production failed to induce protection, showing that protective antibodies are mainly directed against the capsule. Our vaccination protocol also confers increased protection in wild-type mice vaccinated and then treated with cyclophosphamide; an immunosuppressive drug described to strongly increase the susceptibility of mice to A. baumannii infection. Finally, we demonstrate that HK LAC-4 can induce the activation of human monocyte-derived dendritic cells and T lymphocytes from PBMCs of healthy donors, suggesting that it may activate the human adaptive immune system and induce a protective memory response against A. baumannii.ConclusionsOverall, our results demonstrate that administration of HK bacteria can induce protective immunity against A. baumannii in both immuno-competent and immuno-compromised mice and that these HK bacteria can activate the human adaptive immune system.

Neisseria gonorrhoeae is a human-specific pathogen that causes the important sexually transmitted infection, gonorrhoea, an inflammatory condition of the genitourinary tract. The bacterium is closely related to the meningococcus, a leading cause of bacterial meningitis. Both these invasive bacterial species undergo autolysis when in the stationary phase of growth. Autolysis is a form of programmed cell death (PCD) which is part of the life cycle of remarkably few bacteria and poses an evolutionary conundrum as altruistic death provides no obvious benefit for single-celled organisms. Here, we searched for genes present in these 2 invasive species but not in other members of the Neisseria genus. We identified a ~3.4 kb horizontally acquired region, we termed the nap island, which is largely restricted to the gonococcus and meningococcus. The nap island in the gonococcus encodes 3 cationic, bacteriocin-like peptides which have no detectable antimicrobial activity. Instead, the gonococcal Neisseria autolysis peptides (Naps) promote autolytic cell death when bacteria enter the stationary phase of growth. Furthermore, strains lacking the Naps exhibit reduced autolysis in assays of PCD. Expression of Naps is likely to be phase variable, explaining how PCD could have arisen in these important human pathogens. NapC also induces lysis of human cells, so the peptides are likely to have multiple roles during colonisation and disease. The acquisition of the nap island contributed to the emergence of PCD in the gonococcus and meningococcus and potentially to the appearance of invasive disease in Neisseria spp.

Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-μL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8–94.7%) and specificity of 92.4% (95% CI 83.2–97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.

It is assumed that intracellular pathogenic bacteria have to cope with DNA alkylating stress within host cells. Here we use single-cell reporter systems to show that the pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophage infection. Genes encoding direct repair and base-excision repair pathways are required by B. abortus to face this stress in vitro and in a mouse infection model. Among these genes, ogt is found to be under the control of the conserved cell-cycle transcription factor GcrA. Our results highlight that the control of DNA repair in B. abortus displays distinct features that are not present in model organisms such as Escherichia coli.

The α-proteobacteria are a fascinating group of free-living, symbiotic and pathogenic organisms, including the Brucella genus, which is responsible for a worldwide zoonosis. One common feature of α-proteobacteria is the presence of a conserved response regulator called CtrA, first described in the model bacterium Caulobacter crescentus, where it controls gene expression at different stages of the cell cycle. Here, we focus on Brucella abortus and other intracellular α-proteobacteria in order to better assess the potential role of CtrA in the infectious context. Comparative genomic analyses of the CtrA control pathway revealed the conservation of specific modules, as well as the acquisition of new factors during evolution. The comparison of CtrA regulons also suggests that specific clades of α-proteobacteria acquired distinct functions under its control, depending on the essentiality of the transcription factor. Other CtrA-controlled functions, for instance motility and DNA repair, are proposed to be more ancestral. Altogether, these analyses provide an interesting example of the plasticity of a regulation network, subject to the constraints of inherent imperatives such as cell division and the adaptations to diversified environmental niches.

Brucella abortus is a class III zoonotic bacterial pathogen able to survive and replicate inside host cells, including macrophages. Here we report a multidimensional transposon sequencing analysis to identify genes essential for Brucella abortus growth in rich medium and replication in RAW 264.7 macrophages. The construction of a dense transposon mutant library and mapping of 929,769 unique mini-Tn5 insertion sites in the genome allowed identification of 491 essential coding sequences and essential segments in the B. abortus genome. Chromosome II carries a lower proportion (5%) of essential genes than chromosome I (19%), supporting the hypothesis of a recent acquisition of a megaplasmid as the origin of chromosome II. Temporally resolved transposon sequencing analysis as a function of macrophage infection stages identified 79 genes with a specific attenuation phenotype in macrophages, at either 2, 5, or 24 h postinfection, and 86 genes for which the attenuated mutant phenotype correlated with a growth defect on plates. We identified 48 genes required for intracellular growth, including the virB operon, encoding the type IV secretion system, which supports the validity of the screen. The remaining genes encode amino acid and pyrimidine biosynthesis, electron transfer systems, transcriptional regulators, and transporters. In particular, we report the need of an intact pyrimidine nucleotide biosynthesis pathway in order for B. abortus to proliferate inside RAW 264.7 macrophages.

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP-seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution.

ABSTRACT Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Capsular polysaccharide vaccines are available against meningococcal serogroups A, C, W, and Y. More recently two protein-based vaccines, Bexsero and Trumenba, against meningococcal serogroup B strains have been licensed; both vaccines contain meningococcal factor H binding protein (fHbp). fHbp is a surface-exposed lipoprotein that binds the negative complement regulator complement factor H (CFH), thereby inhibiting the alternative pathway of complement activation. Recent analysis of available genomes has indicated that some commensal Neisseria species also contain genes that potentially encode fHbp, although the functions of these genes and how immunization with fHbp-containing vaccines could affect the commensal flora have yet to be established. Here, we show that the commensal species Neisseria cinerea expresses functional fHbp on its surface and that it is responsible for recruitment of CFH by the bacterium. N. cinerea fHbp binds CFH with affinity similar to that of meningococcal fHbp and promotes survival of N. cinerea in human serum. We examined the potential impact of fHbp-containing vaccines on N. cinerea . We found that immunization with Bexsero elicits serum bactericidal activity against N. cinerea , which is primarily directed against fHbp. The shared function of fHbp in N. cinerea and N. meningitidis and cross-reactive responses elicited by Bexsero suggest that the introduction of fHbp-containing vaccines has the potential to affect carriage of N. cinerea and other commensal species.