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Abstract Bacteria adapt to selective pressure in their immediate environment in multiple ways. One mechanism involves the acquisition of independent mutations that disable or modify a key pathway, providing a signature of adaptation via convergent evolution. Extra-intestinal pathogenic Escherichia coli (ExPEC) belonging to sequence type 95 (ST95) represent a global clone frequently associated with severe human infections including acute pyelonephritis, sepsis, and neonatal meningitis. Here, we analysed a publicly available dataset of 613 ST95 genomes and identified a series of loss-of-function mutations that disrupt cellulose production or its modification in 55.3% of strains. We show the inability to produce cellulose significantly enhances ST95 invasive infection in a rat model of neonatal meningitis, leading to the disruption of intestinal barrier integrity in newborn pups and enhanced dissemination to the liver, spleen and brain. Consistent with these observations, disruption of cellulose production in ST95 augmented innate immune signalling and tissue neutrophil infiltration in a mouse model of urinary tract infection. Mutations that disrupt cellulose production were also identified in other virulent ExPEC STs, Shigella and Salmonella, suggesting a correlative association with many Enterobacteriaceae that cause severe human infection. Together, our findings provide an explanation for the emergence of hypervirulent Enterobacteriaceae clones.

Abstract Mucosally active subunit vaccines are an unmet clinical need due to lack of licensed immunostimulants suitable for vaccine antigens. Here, we show that intranasal administration of liposomes incorporating: the Streptococcus pyogenes peptide antigen, J8; diphtheria toxoid as a source of T cell help; and the immunostimulatory glycolipid, 3D(6-acyl) PHAD (PHAD), is able to induce long-lived humoral and cellular immunity. Mice genetically deficient in either mucosal antibodies or total antibodies are protected against S. pyogenes respiratory tract infection. Utilizing IL-17-deficient mice or depleting cellular subsets using antibodies, shows that the cellular responses encompassing, CD4+ T cells, IL-17, macrophages and neutrophils have important functions in vaccine-mediated mucosal immunity. Overall, these data demonstrate the utility of a mucosal vaccine platform to deliver multi-pronged protective responses against a highly virulent pathogen.

Antigenic cell fragments, pathogen-associated molecular patterns, and other immunostimulants in bacterial lysates or extracts may induce local and systemic immune responses in specific and nonspecific paradigms. Based on current knowledge, this review aimed to determine whether bacterial lysate has comparable functions in infectious diseases and cancer treatment. In infectious diseases, including respiratory and urinary tract infections, immune system activation by bacterial lysate can identify and combat pathogens. Commercially available bacterial lysates, including OM-85, Ismigen, Lantigen B, and LW 50020, were effective in children and adults in treating respiratory tract infections, chronic obstructive pulmonary disease, rhinitis, and rhinosinusitis with varying degrees of success. Moreover, OM-89, Uromune, Urovac, Urivac, and ExPEC4V showed therapeutic benefits in controlling urinary tract infections in adults, especially women. Bacterial lysate-based therapeutics are safe, well-tolerated, and have few side effects, making them a good alternative for infectious disease management. Furthermore, a nonspecific immunomodulation by bacterial lysates may stimulate innate immunity, benefiting cancer treatment. “Coley’s vaccine” has been used to treat sarcomas, carcinomas, lymphomas, melanomas, and myelomas with varying outcomes. Later, several similar bacterial lysate-based therapeutics have been developed to treat cancers, including bladder cancer, non-small cell lung cancer, and myeloma; among them, BCG for in situ bladder cancer is well-known. Proinflammatory cytokines, including IL-1, IL-6, IL-12, and TNF-α, may activate bacterial antigen-specific adaptive responses that could restore tumor antigen recognition and response by tumor-specific type 1 helper cells and cytotoxic T cells; therefore, bacterial lysates are worth investigating as a vaccination adjuvants or add-on therapies for several cancers.

Abstract Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is a frequent cause of infections, including bacteraemia and other acute diseases in adults and immunocompromised individuals. We developed a novel system to study GBS within human monocytes to define the co-transcriptome of intracellular GBS (iGBS) and host cells simultaneously using dual RNA-sequencing (RNA-seq) to better define how this pathogen responds to host cells. Using human U937 monocytes and genome-sequenced GBS reference strain 874,391 in antibiotic protection assays we validated a system for dual-RNA seq based on measures of GBS and monocyte viability to ensure that the bacterial and host cell co-transcriptome reflected mainly intracellular (iGBS) rather than extracellular GBS. Elucidation of the co-transcriptome revealed 1119 dysregulated transcripts in iGBS with most genes, including several that encode virulence factors (e.g., scpB, hvgA, ribD, pil2b) exhibiting activation by upregulated expression. Infection with iGBS resulted in significant remodelling of the monocyte transcriptome, with 7587 transcripts differentially expressed including 7040 up-regulated and 547 down-regulated. qPCR confirmed that the most strongly activated genes included sht, encoding Streptococcal Histidine Triad Protein. An isogenic GBS mutant strain deficient in sht revealed a significant effect of this gene on phagocytosis of GBS and survival of the bacteria during systemic infection in mice. Identification of a novel contribution of sht to GBS virulence shows the co-transcriptome responses elucidated in GBS-infected monocytes help to shape the host–pathogen interaction and establish a role for sht in the response of the bacteria to phagocytic uptake. This study provides comprehension of concurrent transcriptional responses that occur in GBS and human monocytes that shape the host–pathogen interaction.

Streptococcus agalactiae, also known as group B Streptococcus, is an important human and animal pathogen. Zinc (Zn) is an essential trace element for normal bacterial physiology but intoxicates bacteria at high concentrations. Molecular systems for Zn detoxification exist in S. agalactiae, however the degree to which Zn detoxification may vary among different S. agalactiae isolates is not clear. We measured resistance to Zn intoxication in a diverse collection of clinical isolates of S. agalactiae by comparing the growth of the bacteria in defined conditions of Zn stress. We found significant differences in the ability of different S. agalactiae isolates to resist Zn intoxication; some strains such as S. agalactiae 18RS21 were able to survive and grow at 3.8-fold higher levels of Zn stress compared to other reference strains such as BM110 (6.4mM vs 1.68mM Zn as inhibitory, respectively). We performed in silico analysis of the available genomes of the S. agalactiae isolates used in this study to examine the sequence of czcD, which encodes an efflux protein for Zn that supports resistance in S. agalactiae. Interestingly, this revealed the presence of a mobile insertion sequence (IS) element, termed IS1381, in the 5′ region of czcD in S. agalactiae strain 834, which was hyper-resistant to Zn intoxication. Interrogating a wider collection of S. agalactiae genomes revealed identical placement of IS1381 in czcD in other isolates from the clonal-complex-19 (CC19) 19 lineage. Collectively, these results show a resistance spectrum among S. agalactiae isolates enables survival in varying degrees of Zn stress, and this phenotypic variability has implications for understanding bacterial survival in metal stress.

Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH), encoded by gapC, is a glycolytic enzyme that is associated with virulence and immune-mediated protection. However, the role of GAPDH in cellular cytokine responses to S. agalactiae, bacterial phagocytosis and colonization of the female reproductive tract, a central host niche, is unknown. We expressed and studied purified recombinant GAPDH (rGAPDH) of S. agalactiae in cytokine elicitation assays with human monocyte-derived macrophage, epithelial cell, and polymorphonuclear leukocyte (PMN) co-culture infection models. We also generated a S. agalactiae mutant that over-expresses GAPDH (oeGAPDH) from gapC using a constitutively active promoter, and analyzed the mutant in murine macrophage antibiotic protection assays and in virulence assays in vivo, using a colonization model that is based on experimental infection of the reproductive tract in female mice. Human cell co-cultures produced interleukin (IL)-1β, IL-6, macrophage inflammatory protein (MIP)-1, tumor necrosis factor (TNF)-α and IL-10 within 24 h of exposure to rGAPDH. PMNs were required for several of these cytokine responses. However, over-expression of GAPDH in S. agalactiae did not significantly affect measures of phagocytic uptake compared to an empty vector control. In contrast, oeGAPDH-S. agalactiae showed a small but statistically significant attenuation for persistence in the reproductive tract of female mice during the chronic phase of infection (10–28 days post-inoculation), relative to the vector control. We conclude that S. agalactiae GAPDH elicits production of multiple cytokines from human cells, and over-expression of GAPDH renders the bacterium more susceptible to host clearance in the female reproductive tract. One-sentence summary: This study shows Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase, an enzyme that functions in glycolysis, gluconeogenesis and virulence, modifies phagocytosis outcomes, including cytokine synthesis, and affects bacterial persistence in the female reproductive tract.

Metals such as copper (Cu) and zinc (Zn) are important trace elements that can affect bacterial cell physiology but can also intoxicate bacteria at high concentrations. Discrete genetic systems for management of Cu and Zn efflux have been described in several bacterial pathogens, including streptococci. However, insight into molecular cross-talk between systems for Cu and Zn management in bacteria that drive metal detoxification, is limited. Here, we describe a biologically consequential cross-system effect of metal management in group B Streptococcus (GBS) governed by the Cu-responsive copY regulator in response to Zn. RNAseq analysis of wild-type (WT) and copY-deficient GBS subjected to metal stress revealed unique transcriptional links between the systems for Cu and Zn detoxification. We show that the Cu-sensing role of CopY extends beyond Cu and enables CopY to regulate Cu and Zn stress responses that effect changes in gene function for central cellular processes, including riboflavin synthesis. CopY also supported GBS intracellular survival in human macrophages and virulence during disseminated infection in mice. In addition, we show a novel role for CovR in modulating GBS resistance to Zn intoxication. Identification of the Zn resistome of GBS using TraDIS revealed a suite of genes essential for GBS growth in metal stress. Several of the genes identified are novel to systems that support bacterial survival in metal stress and represent a diverse set of mechanisms that underpin microbial metal homeostasis during cell stress. Overall, this study reveals a new and important mechanism of cross-system complexity driven by CopY in bacteria to regulate cellular management of metal stress and survival.

ABSTRACT Many antibiotic resistant uropathogenic Escherichia coli (UPEC) strains belong to clones defined by their multilocus sequence type (ST), with ST131 being the most dominant. Although we have a good understanding of resistance development to fluoroquinolones and third-generation cephalosporins by ST131, our understanding of the virulence repertoire that has contributed to its global dissemination is limited. Here we show that the genes encoding Afa/Dr fimbriae, a group of adhesins strongly associated with UPEC that cause gestational pyelonephritis and recurrent cystitis, are found in approximately one third of all ST131 strains. Sequence comparison of the AfaE adhesin protein revealed a unique allelic variant carried by 82.9% of afa-positive ST131 strains. We identify the afa regulatory region as a hotspot for the integration of insertion sequence (IS) elements, all but one of which alter afa transcription. Close investigation demonstrated that the integration of an IS1 element in the afa regulatory region leads to increased expression of Afa/Dr fimbriae, promoting enhanced adhesion to kidney epithelial cells and suggesting a mechanism for altered virulence. Finally, we provide evidence for a more widespread impact of IS1 on ST131 genome evolution, suggesting that IS dynamics contribute to strain level microevolution that impacts ST131 fitness. IMPORTANCE E. coli ST131 is the most common antibiotic resistant UPEC clone associated with human urinary tract and bloodstream infections. Understanding the features of ST131 that have driven its global dissemination remains a critical priority if we are to counter its increasing antibiotic resistance. Here, we utilized a large collection of ST131 isolates to investigate the prevalence, regulation, and function of Afa/Dr fimbriae, a well-characterized UPEC colonization and virulence factor. We show that the afa genes are found frequently in ST131 and demonstrate how the integration of IS elements in the afa regulatory region modulates Afa expression, presenting an example of altered virulence capacity. We also exploit a curated set of ST131 genomes to map the integration of the antibiotic resistance-associated IS1 element in the ST131 pangenome, providing evidence for its widespread impact on ST131 genome evolution.

Streptococcus agalactiae causes neonatal meningitis and can also infect the adult central nervous system (CNS). S. agalactiae can cross the blood-brain barrier but may also reach the CNS via other paths. Several species of bacteria can directly invade the CNS via the olfactory and trigeminal nerves, which extend between the nasal cavity and brain and injury to the nasal epithelium can increase the risk/severity of infection. Preterm birth is associated with increased risk of S. agalactiae infection and with nasogastric tube feeding. The tubes, also used in adults, can cause nasal injuries and may be contaminated with bacteria, including S. agalactiae. We here investigated whether S. agalactiae could invade the CNS after intranasal inoculation in mice. S. agalactiae rapidly infected the olfactory nerve and brain. Methimazole-mediated model of nasal epithelial injury led to increased bacterial load in these tissues, as well as trigeminal nerve infection. S. agalactiae infected and survived intracellularly in cultured olfactory/trigeminal nerve- and brain-derived glia, resulting in cytokine production, with some differences between glial types. Furthermore, a non-capsulated S. agalactiae was used to understand the role of capsule on glial cells interaction. Interestingly, we found that the S. agalactiae capsule significantly altered cytokine and chemokine responses and affected intracellular survival in trigeminal glia. In summary, this study shows that S. agalactiae can infect the CNS via the nose-to-brain path with increased load after epithelial injury, and that the bacteria can survive in glia.

This case study describes how high school students can participate in research, development, and testing of real-life solutions for society’s most intractable problems. In modern warfare, civilians make up most of the casualties, and conflict-affected communities are often isolated and have only limited access to healthcare. Most surviving casualties have limb wounds from injury or surgery, and many of these become infected and require long-term treatment or amputation. In 2020, MedMagLabs and the Queensland Virtual STEM Academy partnered to engage high school students to co-develop and test methods and training resources that empower people in conflict-affected communities to produce medicinal maggots for highly efficacious and affordable wound care. Maggot therapy is the treatment of wounds with living fly larvae to remove dead tissue, to control infection, and to promote wound healing. As opposed to most citizen science, which mainly focuses on data collection and/or educational and awareness-raising outcomes, this project focused on the co-creation of knowledge and the delivery of tangible research outcomes. The measure of its success was the development of end-user friendly medicinal maggot production methods and training resources. The study explains how citizen scientists and researchers collaborated with one another to achieve this objective. Project execution was largely in line with The Ten Principles of Citizen Science. Further review of project outcomes and self-reflection by the research team highlight important lessons for such collaborative studies, which have been summarised in five recommendations specifically relating to research collaborations with schools and student citizen scientists.