Neisseria gonorrhoeae is a common Gram-negative pathogen with increasing resistance to all recommended antibiotics. There is a critical need to improve the efficiency of the antibiotic hit discovery process to replenish the drug development pipeline. Here, we show that deep learning models can augment high-throughput screens to identify readily available molecules with narrow-spectrum activity against difficult-to-treat strains of N. gonorrhoeae. We phenotypically tested 38,650 small molecules for N. gonorrhoeae growth inhibition to train a predictive graph neural network (GNN) model. We benchmarked the model's performance against other architectures, including a large language model, and found that GNNs more accurately identify active, drug-like molecules that are structurally distinct from the training set and known antibiotics. Using the model to virtually screen ~6 million compounds, we identified 213 compounds for experimental validation and found that 83 (39%) inhibited N. gonorrhoeae growth. Two of these compounds were structurally dissimilar to existing antibiotics, maintained potency against multidrug-resistant N. gonorrhoeae strains in vitro, exhibited promising selectivity indices, and were rapidly bactericidal with low frequencies of resistance. Proteomic studies revealed their distinct mechanisms of action, with one compound targeting alanine racemase, an enzyme involved in the essential process of peptidoglycan synthesis. Furthermore, the compounds showed early promise in reducing N. gonorrhoeae titers in a human vagina-on-a-chip infection model and a mouse vaginal infection model. Our work establishes the deep learning-enabled discovery of selective antibacterial compounds against N. gonorrhoeae as a much-needed hit discovery tool to address the growing crisis of antimicrobial resistance for this pathogen.

The antimicrobial resistance crisis necessitates structurally distinct antibiotics. While deep learning approaches can identify antibacterial compounds from existing libraries, structural novelty remains limited. Here, we developed a generative artificial intelligence framework for designing de novo antibiotics through two approaches: a fragment-based method to comprehensively screen >107 chemical fragments in silico against Neisseria gonorrhoeae or Staphylococcus aureus, subsequently expanding promising fragments, and an unconstrained de novo compound generation, each using genetic algorithms and variational autoencoders. Of 24 synthesized compounds, seven demonstrated selective antibacterial activity. Two lead compounds exhibited bactericidal efficacy against multidrug-resistant isolates with distinct mechanisms of action and reduced bacterial burden in vivo in mouse models of N. gonorrhoeae vaginal infection and methicillin-resistant S. aureus skin infection. We further validated structural analogs for both compound classes as antibacterial. Our approach enables the generative deep-learning-guided design of de novo antibiotics, providing a platform for mapping uncharted regions of chemical space.

Degradation of immunoglobulin (Ig) represents an important bacterial immune evasion strategy. For mucosal colonization, degradation of IgA is of particular importance, and many bacteria secrete specific IgA proteases that typically target the extended hinge region of IgA1. Such a specialized IgA protease has not yet been reported in Group A Streptococcus (GAS), despite its ability to successfully colonize human mucosal surfaces. In this study, we focused on the cysteine protease SpeB secreted by GAS and analyzed the interaction of SpeB with IgA. Assays using bacterial supernatants from wild-type and speB-deficient isogenic mutant strains, as well as recombinant SpeB, showed a SpeB-dependent IgA-modifying activity. SpeB resulted in the degradation of multimeric IgA, including the dimeric form, which was most notable in IgA2. The modification products were smaller in size than the heavy chain, suggesting a modification different from the classical hinge cleavage. Mass spectrometry analysis and glycosylation profiles indicated a putative cleavage in the C-terminal region, affecting the tailpiece and resulting in the loss of higher molecular weight multimeric/dimeric forms of IgA. Given the importance of dimeric IgA at mucosal surfaces, future studies are warranted to address whether IgA modification by SpeB represents a GAS immune evasion mechanism at this site.IMPORTANCEGroup A Streptococcus (GAS) is an important human pathogen with the ability to efficiently colonize mucosal surfaces and cause a wide spectrum of diseases ranging from pharyngotonsillitis to severe invasive infections or post-streptococcal sequelae. Immunoglobulins (Ig), in particular IgA, are critical effector molecules in the defense against pathogen colonization at mucosal surfaces. In this study, we focused on the cysteine protease SpeB, secreted by GAS, and investigated its interaction with human IgA. We report a SpeB-dependent IgA modification that involved the loss of multimeric/dimeric forms of IgA, predominantly affecting IgA2. The putative modification region is the C-terminus of IgA, which differs from the cleavage site of specialized IgA proteases targeting the hinge region. These findings suggest that IgA modification by SpeB might represent an immune evasion strategy utilized by GAS to colonize human mucosal tissue.

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.

Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays.

Objectives Meningitis is a medical emergency, and it is crucial to diagnose it accurately and promptly in order to manage patients effectively. It would, therefore, be essential to introduce and have fast, accurate, and user-friendly methods to determine the cause of these infections. This study aimed to demonstrate a potentially cost-effective new approach for detecting meningitis using a paper-based vertical flow microarray, which could be useful in settings with limited resources. Methods We describe a multiplex paper microarray for detecting Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, and Salmonella spp. by the passive vertical flow of PCR-amplified clinical samples. A multibiotinylated amplicon was obtained as a product of PCR in the presence of both a biotinylated primer and biotin-11-dUTP. An enhancement step based on an enzyme-free gold enhancement protocol was also used to facilitate visual detection. Results This study showed that the vertical flow microarray (previously evaluated for one pathogen) can discriminately detect the amplification results down to the 102 copies of DNA limit for four meningitis pathogens in a multiplexed set-up. The study further demonstrated the ability of this device and setup to detect three of the four pathogens from clinical biosamples. Discussion This study demonstrated the capacity of a vertical flow microarray device to detect amplification products for four prevalent meningitis pathogens in a multiplex format. The vertical flow microarray demonstrated consistent visualization of the expected gene amplification results; however, indicating limitations in the pre- and amplification steps. This study highlights the potential of this multiplexing method for diagnosing meningitis and other syndromic diseases caused by various pathogens, especially in resource-limited areas.

In this Journal Club, Edmund Loh explores the pioneering work of Jun-Ichi Tomizawa and colleagues, which uncovers the importance of a small RNA in plasmid replication and elucidates its mechanism of action.

The study of bacterial gene expression during infection provides vital information for researchers to understand bacterial pathogenesis and infection. The ability to obtain clean and undegraded RNA could be challenging and daunting and remains the most crucial experimental step prior to downstream analyses, such as Northern blotting, quantitative PCR (qPCR), and RNA-seq.This chapter describe two methods (acid guanidinium thiocyanate (TRIzol) phenol-chloroform and hot phenol) commonly used to isolate total bacterial RNA and are suitable for both Gram-positive and Gram-negative bacteria. Procedures such as RNA quantification and DNase treatment are also included to ensure amount and quality of the RNA samples. The second part of the chapter includes a method used to analyze bacterial gene expression (Northern blotting), two methods to generate radioactive probes, as well as target detection using a phosphorimager.

Pathogenic bacteria have evolved to sense their surrounding environments and regulate their gene expression to evade host immune defences and cause disease. RNA-mediated gene expression offers a fast and energy efficient alternative to conventional transcription factors. A myriad of regulatory RNAs have been identified, especially in pathogenic bacteria. However, whether these RNAs partake in disease remains largely unexplored. Here, we review current knowledge of regulatory RNAs in human-adapted upper respiratory tract pathogens. We propose that bacterial regulatory RNAs could play important roles in disease. Elucidating the function of regulatory RNAs and identifying polymorphisms among disease isolates would provide valuable insight into their pathogeneses. Finally, we discuss the outstanding issues of regulatory RNAs in research and their applications as drug targets, therapeutics, and in providing diagnostic information predictive of disease prognosis.

Neisseria meningitidis (meningococcus) is a human-restricted bacterium and a common coloniser of the nasopharynx. The bacterium is often harmless, but can in rare cases cause life-threatening meningitis and sepsis, commonly referred to as invasive meningococcal disease (IMD). Because of the rapid onset of disease and the risk of severe morbidity and mortality, antibiotics are administered promptly to patients where there is suspicion of IMD. Although vaccination programmes against the meningococcus have substantially reduced IMD worldwide, disease burden remains a challenge, particularly in low-income and middle-income countries.1Bizri AR Althaqafi A Kaabi N Obeidat N Al Akoury N Haridy H The burden of invasive vaccine-preventable diseases in adults in the Middle East and North Africa (MENA) region.Infect Dis Ther. 2021; 10: 663-685Crossref PubMed Scopus (2) Google ScholarThe surveillance network for IMD in Europe started in 1999 as part of the Invasive Bacterial Infections Surveillance project. The aims of the network are to improve the epidemiological information on IMD in Europe, to characterise meningococcal isolates, and to foster international collaborations. This project facilitated the complete genome mapping of two meningococcal strains in 2000.2Tettelin H Saunders NJ Heidelberg J et al.Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.Science. 2000; 287: 1809-1815Crossref PubMed Scopus (953) Google Scholar, 3Parkhill J Achtman M James KD et al.Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491.Nature. 2000; 404: 502-506Crossref PubMed Scopus (610) Google Scholar Since 2009, the Meningitis Research Foundation further commissioned the creation of a publicly available online library of the genomes of all meningococcal disease isolates in the UK, pioneering the public databases for molecular typing and microbial genome diversity (PubMLST).4Jolley KA Bray JE Maiden MCJ Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications.Wellcome Open Res. 2018; 3: 124Crossref PubMed Scopus (760) Google Scholar With the rapid advancement of next-generation sequencing, whole-genome sequencing has now become the gold standard in outbreak investigation and surveillance of IMD in many countries. Generated whole-genome sequencing data are often annotated and deposited in the meningococcal PubMLST.Although there is an extensive meningococcal database, its usage in scientific research has been scarce. The meningococcus has a few tested animal models but they are all only specifically for unique virulence factors and there is no suitable animal model to investigate IMD resembling the human body, which further hinders the investigation process. Publications have highlighted that comparative molecular epidemiological investigation utilising the PubMLST could provide valuable information in assisting the identification of novel meningococcal virulence factors and their association with IMD.The mucosal membranes in the nasopharynx that the meningococcus colonises are often replete with secretory IgA. Meningococci can degrade these IgA using their IgA1-specific serine protease. Our study has shown that the IgA1 serine protease could further facilitate meningococcal immune evasion by opposing serum IgG3.5Spoerry C Karlsson J Aschtgen MS Loh E Neisseria meningitidis IgA1-specific serine protease exhibits novel cleavage activity against IgG3.Virulence. 2021; 12: 389-403Crossref PubMed Scopus (4) Google Scholar Through molecular characterisation, it was shown that meningococcal IgA1-specific serine protease of cleavage type 1 degrades both IgG3 and IgA, whereas cleavage type 2 only degrades IgA. Comparative epidemiological analysis of more than 7000 clinical meningococcal isolates from the PubMLST shows a significantly higher proportion of cleavage type 1 among isolates from invasive cases compared with carrier cases.The meningococcus has several other strategies to evade complement-mediated killing, such as the production of a polysaccharide capsule and surface-exposed Factor H binding protein (fHbp). Both of these factors are major components of the current meningococcal vaccines (Menveo, a quadrivalent polysaccharide conjugate vaccine; and Bexsero, a recombinant protein vaccine for Meningitis B). The expressions of both virulence factors are temperature dependent and are mediated by regulatory RNAs.6Loh E Kugelberg E Tracy A et al.Temperature triggers immune evasion by Neisseria meningitidis.Nature. 2013; 502: 237-240Crossref PubMed Scopus (101) Google Scholar Our study further identified sequence variants within a regulatory RNA that induces hypercapsulation phenotype that confers better protection against complement-mediated killing.7Karlsson J Eichner H Andersson C Jacobsson S Loh E Novel hypercapsulation RNA thermosensor variants in Neisseria meningitidis and their association with invasive meningococcal disease: a genetic and phenotypic investigation and molecular epidemiological study.Lancet Microbe. 2020; 1: e319-e327Summary Full Text Full Text PDF PubMed Scopus (7) Google Scholar Through comparative molecular epidemiological analysis of isolates from the PubMLST, we showed that hypercapsulated variants have higher serum survival and are almost two-times more common in IMD than in carrier isolates. Another study published in 2021 identified and characterised specific polymorphisms in the main regulatory regions of the fHbp gene that are driving fHbp production. By comparing whole-genome sequencing of almost 6000 clinical meningococcal isolates from the PubMLST, the authors discovered that isolates that produce more fHbp were significantly increased in IMD compared with the carrier state.8Spinsanti M Brignoli T Bodini M et al.Deconvolution of intergenic polymorphisms determining high expression of Factor H binding protein in meningococcus and their association with invasive disease.PLoS Pathog. 2021; 17e1009461Crossref PubMed Google ScholarComparative molecular epidemiology utilised in these studies has shown specific meningococcal virulence factors with an augmented propensity in IMD. However, due to a low availability of whole-genome sequencing from carrier isolates within the PubMLST, these studies were not able to assert stronger causation between the virulence factors of carrier and invasive isolates. Considering the ongoing COVID-19 pandemic, the implementation of lockdown policy has had a positive impact on the epidemiology of IMD. An international surveillance study published in 2021 has shown that there has been a significant reduction in the incidence of invasive respiratory disease including IMD during the COVID-19 pandemic.9Brueggemann AB Jansen van Rensburg MJ Shaw D et al.Changes in the incidence of invasive disease due to Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis during the COVID-19 pandemic in 26 countries and territories in the Invasive Respiratory Infection Surveillance Initiative: a prospective analysis of surveillance data.Lancet Digit Health. 2021; 3: e360-e370Summary Full Text Full Text PDF PubMed Scopus (86) Google Scholar Another case study has shown that a patient suffered IMD and concurrently showed positive RT-PCR for SARS-CoV-2.10Gallacher SD Seaton A Meningococcal meningitis and COVID-19 co-infection.BMJ Case Rep. 2020; 13e237366Crossref PubMed Scopus (14) Google Scholar However, the meningococcus co-infection with SARS-CoV-2 remains unclear and requires further investigation.Another potential positive outcome from the COVID-19 pandemic is a renewed public interest in infectious disease research. This increased interest could encourage participation in carrier studies of the meningococcus and create a larger pool of carrier isolate whole-genome sequencing, which would increase the overall capability of databases like PubMLST in finding new important virulence factors related to IMD. With international collaborations among national surveillance centres, epidemiologists, clinicians, and molecular researchers, the identification and characterisation of meningococcal virulence factors and potential diagnostic and therapeutic tools could be made, ultimately preventing and creating better treatments for IMD. Neisseria meningitidis (meningococcus) is a human-restricted bacterium and a common coloniser of the nasopharynx. The bacterium is often harmless, but can in rare cases cause life-threatening meningitis and sepsis, commonly referred to as invasive meningococcal disease (IMD). Because of the rapid onset of disease and the risk of severe morbidity and mortality, antibiotics are administered promptly to patients where there is suspicion of IMD. Although vaccination programmes against the meningococcus have substantially reduced IMD worldwide, disease burden remains a challenge, particularly in low-income and middle-income countries.1Bizri AR Althaqafi A Kaabi N Obeidat N Al Akoury N Haridy H The burden of invasive vaccine-preventable diseases in adults in the Middle East and North Africa (MENA) region.Infect Dis Ther. 2021; 10: 663-685Crossref PubMed Scopus (2) Google Scholar The surveillance network for IMD in Europe started in 1999 as part of the Invasive Bacterial Infections Surveillance project. The aims of the network are to improve the epidemiological information on IMD in Europe, to characterise meningococcal isolates, and to foster international collaborations. This project facilitated the complete genome mapping of two meningococcal strains in 2000.2Tettelin H Saunders NJ Heidelberg J et al.Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.Science. 2000; 287: 1809-1815Crossref PubMed Scopus (953) Google Scholar, 3Parkhill J Achtman M James KD et al.Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491.Nature. 2000; 404: 502-506Crossref PubMed Scopus (610) Google Scholar Since 2009, the Meningitis Research Foundation further commissioned the creation of a publicly available online library of the genomes of all meningococcal disease isolates in the UK, pioneering the public databases for molecular typing and microbial genome diversity (PubMLST).4Jolley KA Bray JE Maiden MCJ Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications.Wellcome Open Res. 2018; 3: 124Crossref PubMed Scopus (760) Google Scholar With the rapid advancement of next-generation sequencing, whole-genome sequencing has now become the gold standard in outbreak investigation and surveillance of IMD in many countries. Generated whole-genome sequencing data are often annotated and deposited in the meningococcal PubMLST. Although there is an extensive meningococcal database, its usage in scientific research has been scarce. The meningococcus has a few tested animal models but they are all only specifically for unique virulence factors and there is no suitable animal model to investigate IMD resembling the human body, which further hinders the investigation process. Publications have highlighted that comparative molecular epidemiological investigation utilising the PubMLST could provide valuable information in assisting the identification of novel meningococcal virulence factors and their association with IMD. The mucosal membranes in the nasopharynx that the meningococcus colonises are often replete with secretory IgA. Meningococci can degrade these IgA using their IgA1-specific serine protease. Our study has shown that the IgA1 serine protease could further facilitate meningococcal immune evasion by opposing serum IgG3.5Spoerry C Karlsson J Aschtgen MS Loh E Neisseria meningitidis IgA1-specific serine protease exhibits novel cleavage activity against IgG3.Virulence. 2021; 12: 389-403Crossref PubMed Scopus (4) Google Scholar Through molecular characterisation, it was shown that meningococcal IgA1-specific serine protease of cleavage type 1 degrades both IgG3 and IgA, whereas cleavage type 2 only degrades IgA. Comparative epidemiological analysis of more than 7000 clinical meningococcal isolates from the PubMLST shows a significantly higher proportion of cleavage type 1 among isolates from invasive cases compared with carrier cases. The meningococcus has several other strategies to evade complement-mediated killing, such as the production of a polysaccharide capsule and surface-exposed Factor H binding protein (fHbp). Both of these factors are major components of the current meningococcal vaccines (Menveo, a quadrivalent polysaccharide conjugate vaccine; and Bexsero, a recombinant protein vaccine for Meningitis B). The expressions of both virulence factors are temperature dependent and are mediated by regulatory RNAs.6Loh E Kugelberg E Tracy A et al.Temperature triggers immune evasion by Neisseria meningitidis.Nature. 2013; 502: 237-240Crossref PubMed Scopus (101) Google Scholar Our study further identified sequence variants within a regulatory RNA that induces hypercapsulation phenotype that confers better protection against complement-mediated killing.7Karlsson J Eichner H Andersson C Jacobsson S Loh E Novel hypercapsulation RNA thermosensor variants in Neisseria meningitidis and their association with invasive meningococcal disease: a genetic and phenotypic investigation and molecular epidemiological study.Lancet Microbe. 2020; 1: e319-e327Summary Full Text Full Text PDF PubMed Scopus (7) Google Scholar Through comparative molecular epidemiological analysis of isolates from the PubMLST, we showed that hypercapsulated variants have higher serum survival and are almost two-times more common in IMD than in carrier isolates. Another study published in 2021 identified and characterised specific polymorphisms in the main regulatory regions of the fHbp gene that are driving fHbp production. By comparing whole-genome sequencing of almost 6000 clinical meningococcal isolates from the PubMLST, the authors discovered that isolates that produce more fHbp were significantly increased in IMD compared with the carrier state.8Spinsanti M Brignoli T Bodini M et al.Deconvolution of intergenic polymorphisms determining high expression of Factor H binding protein in meningococcus and their association with invasive disease.PLoS Pathog. 2021; 17e1009461Crossref PubMed Google Scholar Comparative molecular epidemiology utilised in these studies has shown specific meningococcal virulence factors with an augmented propensity in IMD. However, due to a low availability of whole-genome sequencing from carrier isolates within the PubMLST, these studies were not able to assert stronger causation between the virulence factors of carrier and invasive isolates. Considering the ongoing COVID-19 pandemic, the implementation of lockdown policy has had a positive impact on the epidemiology of IMD. An international surveillance study published in 2021 has shown that there has been a significant reduction in the incidence of invasive respiratory disease including IMD during the COVID-19 pandemic.9Brueggemann AB Jansen van Rensburg MJ Shaw D et al.Changes in the incidence of invasive disease due to Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis during the COVID-19 pandemic in 26 countries and territories in the Invasive Respiratory Infection Surveillance Initiative: a prospective analysis of surveillance data.Lancet Digit Health. 2021; 3: e360-e370Summary Full Text Full Text PDF PubMed Scopus (86) Google Scholar Another case study has shown that a patient suffered IMD and concurrently showed positive RT-PCR for SARS-CoV-2.10Gallacher SD Seaton A Meningococcal meningitis and COVID-19 co-infection.BMJ Case Rep. 2020; 13e237366Crossref PubMed Scopus (14) Google Scholar However, the meningococcus co-infection with SARS-CoV-2 remains unclear and requires further investigation. Another potential positive outcome from the COVID-19 pandemic is a renewed public interest in infectious disease research. This increased interest could encourage participation in carrier studies of the meningococcus and create a larger pool of carrier isolate whole-genome sequencing, which would increase the overall capability of databases like PubMLST in finding new important virulence factors related to IMD. With international collaborations among national surveillance centres, epidemiologists, clinicians, and molecular researchers, the identification and characterisation of meningococcal virulence factors and potential diagnostic and therapeutic tools could be made, ultimately preventing and creating better treatments for IMD. We declare no competing interests.

Bacterial meningitis is a major cause of death and disability in children worldwide. Two human restricted respiratory pathogens, Streptococcus pneumoniae and Haemophilus influenzae , are the major causative agents of bacterial meningitis, attributing to 200,000 deaths annually. These pathogens are often part of the nasopharyngeal microflora of healthy carriers. However, what factors elicit them to disseminate and cause invasive diseases, remain unknown. Elevated temperature and fever are hallmarks of inflammation triggered by infections and can act as warning signals to pathogens. Here, we investigate whether these respiratory pathogens can sense environmental temperature to evade host complement-mediated killing. We show that productions of two vital virulence factors and vaccine components, the polysaccharide capsules and factor H binding proteins, are temperature dependent, thus influencing serum/opsonophagocytic killing of the bacteria. We identify and characterise four novel RNA thermosensors in S . pneumoniae and H . influenzae , responsible for capsular biosynthesis and production of factor H binding proteins. Our data suggest that these bacteria might have independently co-evolved thermosensing abilities with different RNA sequences but distinct secondary structures to evade the immune system.

Neisseria meningitidis (meningococcus) is a common bacterial colonizer of the human nasopharynx but can occasionally cause very severe systemic infections with rapid onset. Meningococci are able to degrade IgA encountered during colonization of mucosal membranes using their IgA1-specific serine protease. During systemic infection, specific IgG can induce complement-mediated lysis of the bacterium. However, meningococcal immune evasion mechanisms in thwarting IgG remain undescribed. In this study, we report for the first time that the meningococcal IgA1-specific serine protease is able to degrade IgG3 in addition to IgA. The IgG3 heavy chain is specifically cleaved in the lower hinge region thereby separating the antigen binding part from its effector binding part. Through molecular characterization, we demonstrate that meningococcal IgA1-specific serine protease of cleavage type 1 degrades both IgG3 and IgA, whereas cleavage type 2 only degrades IgA. Epidemiological analysis of 7581 clinical meningococcal isolates shows a significant higher proportion of cleavage type 1 among isolates from invasive cases compared to carrier cases, regardless of serogroup. Notably, serogroup W cc11 which is an increasing cause of invasive meningococcal disease globally harbors almost exclusively cleavage type 1 protease. Our study also shows an increasing prevalence of meningococcal isolates encoding IgA1P cleavage type 1 compared to cleavage type 2 during the observed decade (2010-2019). Altogether, our work describes a novel mechanism of IgG3 degradation by meningococci and its association to invasive meningococcal disease.

Increasing evidence has demonstrated that regulatory RNA elements such as riboswitches (RS) play a pivotal role in the fine-tuning of bacterial gene expression. In this study, we investigated and characterized a novel transcriptional thiamine pyrophosphate (TPP) RS in the obligate human pathogen N. meningitidis MC58 (serogroup B). This RS is located in the 5´ untranslated region upstream of thiC gene, encoding a protein involved in TPP biosynthesis, an essential cofactor for all living beings. Primer extension revealed the transcriptional start site of thiC. Northern blot analysis of thiC mRNA and reporter gene studies confirmed the presence of an active TPP-sensing RS. Expression patterns of the wild-type RS and site-specific mutants showed that it is an OFF switch that controls transcription elongation of thiC mRNA. Interestingly, the regulatory mechanism of the meningococcal thiC RS resembles the Gram-positive Bacillus subtilis thiC RS rather than the Gram-negative Escherichia coli thiC RS. Therefore, the meningococcal thiC RS represents a rare example of transcriptional RS in a Gram-negative bacterium. We further observed that the RS is actively involved in modulating gene expression in response to different growth media and to supplemented bacterial and eukaryotic cell lysates as possible sources of nutrients in the nasopharynx. Our results suggest that RS-mediated gene regulation could influence meningococcal fitness, through the fine-tuning of biosynthesis and scavenging of nutrients and cofactors, such as thiamine.

Background Neisseria meningitidis is the causative agent of invasive meningococcal disease and the polysaccharide capsule is one of its major virulence factors. Biosynthesis of the meningococcal capsule is controlled by an RNA thermosensor (RNAT) in the 5'-untranslated region (5'-UTR) of the cssA gene. The function of the RNAT depends on an 8-bp tandem repeat configuration. We aimed to identify and characterise novel RNATs in meningococcal isolates responsible for regulating capsule production. Methods We investigated the allele igr_up_NEIS0055, containing the 5'-UTR of the cssA gene, in clinical meningococcal isolates for which whole-genome sequences are available on the Neisseria PubMLST database and that were isolated in Europe between Jan 1, 2010, and Dec 31, 2018. Eight isolates with different RNAT tandem repeat configurations were selected for genetic and phenotypic studies. The thermosensing capability of the RNAT and capsule production was tested with immunoblots. Bacterial survival by capsule protection was assessed with a human serum stress assay and capsule interference with bacterial cell adhesion was evaluated with a bacterial adhesion assay. The dataset of RNAT configurations was analysed for an association with invasive meningococcal disease, and was stratified to visualise the distribution of RNAT configurations within the meningococcal population. Findings Our search of PubMLST identified 112 alleles for the igr_up_NEIS0055 locus and 7013 N meningitidis isolates. Five novel RNAT tandem repeat configurations were identified and eight RNAT tandem repeat configurations, ranging from 1 x 8-bp up to 8 x 8-bp, were characterised. The disrupted RNATs (1 x 8-bp and 3 x 8-bp to 8 x 8-bp) confer upregulated CssA expression and increased capsule production compared with the native 2 x 8-bp configuration, resulting in a hypercapsulation phenotype. Increased capsule production was associated with higher survival rates in up to 25% human serum. The prevalence of a disrupted RNAT resulting in hypercapsulation was almost twice as high in invasive meningococcal disease isolates compared with carrier isolates. Disrupted RNATs were especially attributed to isolates of capsule group B and C, and clonal complexes 23, 32, 213, and 269. Hypercapsulation in one isolate led to lower adhesion onto pharyngeal cells compared with a similar isolate with low capsule production. Interpretation Six non-canonical RNAT tandem repeat variants (3 x 8-bp to 8 x 8-bp) were identified in the igr_up_NEIS0055 locus of N meningitidis that induce a hypercapsulation phenotype, thus providing the meningococci with better protection against host complement-mediated killing than does the native RNAT (2 x 8-bp). Further research is warranted to strengthen the association between hypercapsulation and the progression of invasive meningococcal disease, and to investigate the role of regulatory RNAs in meningococcal virulence and as potential markers for disease progression. Copyright (C) 2020 The Author(s). Published by Elsevier Ltd.

Background A timely differential diagnostic is essential to identify the etiology of central nervous system (CNS) infections in children, in order to facilitate targeted treatment, manage patients, and improve clinical outcome. Objective The Pediatric Infection-Point-of-Care (PI-POC) trial is investigating novel methods to improve and strengthen the differential diagnostics of suspected childhood CNS infections in low-income health systems such as those in Southwestern Uganda. This will be achieved by evaluating (1) a novel DNA-based diagnostic assay for CNS infections, (2) a commercially available multiplex PCR-based meningitis/encephalitis (ME) panel for clinical use in a facility-limited laboratory setting, (3) proteomics profiling of blood from children with severe CNS infection as compared to outpatient controls with fever yet not severely ill, and (4) Myxovirus resistance protein A (MxA) as a biomarker in blood for viral CNS infection. Further changes in the etiology of childhood CNS infections after the introduction of the pneumococcal conjugate vaccine against Streptococcus pneumoniae will be investigated. In addition, the carriage and invasive rate of Neisseria meningitidis will be recorded and serotyped, and the expression of its major virulence factor (polysaccharide capsule) will be investigated. Methods The PI-POC trial is a prospective observational study of children including newborns up to 12 years of age with clinical features of CNS infection, and age-/sex-matched outpatient controls with fever yet not severely ill. Participants are recruited at 2 Pediatric clinics in Mbarara, Uganda. Cerebrospinal fluid (for cases only), blood, and nasopharyngeal (NP) swabs (for both cases and controls) sampled at both clinics are analyzed at the Epicentre Research Laboratory through gold-standard methods for CNS infection diagnosis (microscopy, biochemistry, and culture) and a commercially available ME panel for multiplex PCR analyses of the cerebrospinal fluid. An additional blood sample from cases is collected on day 3 after admission. After initial clinical analyses in Mbarara, samples will be transported to Stockholm, Sweden for (1) validation analyses of a novel nucleic acid–based POC test, (2) biomarker research, and (3) serotyping and molecular characterization of S. pneumoniae and N. meningitidis. Results A pilot study was performed from January to April 2019. The PI-POC trial enrollment of patients begun in April 2019 and will continue until September 2020, to include up to 300 cases and controls. Preliminary results from the PI-POC study are expected by the end of 2020. Conclusions The findings from the PI-POC study can potentially facilitate rapid etiological diagnosis of CNS infections in low-resource settings and allow for novel methods for determination of the severity of CNS infection in such environment. Trial Registration ClinicalTrials.gov NCT03900091; https://clinicaltrials.gov/ct2/show/NCT03900091 International Registered Report Identifier (IRRID) DERR1-10.2196/21430

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa. This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains. IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection.

Nanoparticles exhibit potential as drug carriers in biomedicine due to their high surface-to-volume ratio that allows for facile drug loading. Nanosized drug delivery systems have been proposed for the delivery of biologics facilitating their transport across epithelial layers and maintaining their stability against proteolytic degradation. Here, we capitalize on a nanomanufacturing process famous for its scalability and reproducibility, flame spray pyrolysis, and produce calcium phosphate (CaP) nanoparticles with tailored properties. The as-prepared nanoparticles are loaded with bovine serum albumin (model protein) and bradykinin (model peptide) by physisorption and the physicochemical parameters influencing their loading capacity are investigated. Furthermore, we implement the developed protocol by formulating CaP nanoparticles loaded with the LL-37 antimicrobial peptide, which is a biological drug currently involved in clinical trials. High loading values along with high reproducibility are achieved. Moreover, it is shown that CaP nanoparticles protect LL-37 from proteolysis in vitro. We also demonstrate that LL-37 retains its antimicrobial activity against Escherichia coli and Streptococcus pneumoniae when loaded on nanoparticles in vitro. Therefore, we highlight the potential of nanocarriers for optimization of the therapeutic profile of existing and emerging biological drugs.

Bacterial meningitis is a major cause of death and disability in children worldwide. Two human restricted pathogens, Streptococcus pneumoniae and Haemophilus influenzae , are the major causative agents of bacterial meningitis, attributing to 200,000 deaths annually. These pathogens are often part of the nasopharyngeal microflora of healthy carriers. However, what factors elicit them to disseminate and cause invasive diseases remain unknown. Elevated temperature and fever are hallmarks of inflammation triggered by infections and can act as warning signal to these pathogens. Here, we investigate whether these pathogens could sense environmental temperature to evade host complement-mediated killing. We show that expression of two vital virulence factors and vaccine components, the capsule and factor H binding proteins, are temperature dependent. We identify and characterize four novel RNA thermosensors in S. pneumoniae and H. influenzae within their 5′-untranslated regions of genes, responsible for capsular biosynthesis and production of factor H binding proteins. Our data further demonstrate that these pathogens have co-evolved thermosensing abilities independently with unique RNA sequences, but distinct secondary structures, to evade the human immune system. Author Summary Streptococcus pneumoniae and Haemophilus influenzae are bacteria that reside in the upper respiratory tract. This harmless colonization may progress to severe and often lethal septicaemia and meningitis, but molecular mechanisms that control why these pathogens invade the circulatory system remain largely unknown. Here we show that both S. pneumoniae and H. influenzae can evade complement killing by sensing the temperature of the host. We identify and characterize four novel RNA thermosensors in S. pneumoniae and H. influenzae within their respective 5′-untranslated regions of genes, influencing capsular biosynthesis and production of factor H binding proteins. Moreover, we show that these RNA thermosensors evolved independently with exclusive unique RNA sequences to sense the temperature in the nasopharynx and in other body sites to avoid immune killing. Our finding that regulatory RNA senses temperatures and directly regulate expression of two important virulence factors and vaccine components of S. pneumoniae and H. influenzae , is most important for our understanding of bacterial pathogenesis and for vaccine development. Our work could pave the way for similar studies in other important bacterial pathogens and enables clinicians and microbiologists to adjust their diagnostic techniques, and treatments to best fit the condition of the patients.

The strictly human pathogen Neisseria meningitidis is a commensal bacterium but can occasionally turn lethal causing septicaemia and meningitis. The mechanisms of how the meningococcus shifts to invasive infection remain poorly understood. Here we demonstrate that an eight base-pair tandem repeat deletion in the 5′-untranslated region of the polysaccharide capsular biosynthesis operon results in a hypercapsulation phenotype in clinical isolates. The increased capsule production significantly improves the bacterium survival in human serum while impairing its ability to adhere and colonise human pharyngeal cells. Among 4501 reported meningococcal cases in Europe from 2010-2018, the loss of an eight base-pair tandem repeat is three times more prevalent in invasive isolates (16.3%) compared to carrier isolates (5.1%). Combined results indicate that polymorphisms in this regulatory RNA contributes to meningococcal virulence.