A conserved antigen induces respiratory Th17-mediated broad serotype protection against pneumococcal superinfection.

Several vaccines targeting bacterial pathogens show reduced efficacy upon concurrent viral infection, indicating that a new vaccinology approach is required. To identify antigens for the human pathogen Streptococcus pneumoniae that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the conserved lafB gene as crucial for virulence. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against S. pneumoniae serotypes 2, 15A, and 24F in a murine model. In contrast to standard capsule-based vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, indicating LafB antigenicity in humans. Collectively, these findings present a universal pneumococcal vaccine antigen that remains effective following influenza infection.

Klebsiella pneumoniae OmpR facilitates lung infection through transcriptional regulation of key virulence factors.

IMPORTANCE: Bacteria use two-component regulatory systems (TCSs) to adapt to changes in their environment by changing their gene expression. In this study, we show that the EnvZ/OmpR TCS of the clinically relevant opportunistic pathogen Klebsiella pneumoniae plays an important role in successfully establishing lung infection and virulence. In addition, we elucidate the K. pneumoniae OmpR regulon within the host. This work suggests that K. pneumoniae OmpR might be a promising target for innovative anti-infectives.

Deep Divergence and Genomic Diversification of Gut Symbionts of Neotropical Stingless Bees.

Social bees harbor conserved gut microbiotas that may have been acquired in a common ancestor of social bees and subsequently codiversified with their hosts. However, most of this knowledge is based on studies on the gut microbiotas of honey bees and bumblebees. Much less is known about the gut microbiotas of the third and most diverse group of social bees, the stingless bees. Specifically, the absence of genomic data from their microbiotas presents an important knowledge gap in understanding the evolution and functional diversity of the social bee microbiota. Here, we combined community profiling with culturing and genome sequencing of gut bacteria from six neotropical stingless bee species from Brazil. Phylogenomic analyses show that most stingless bee gut isolates form deep-branching sister clades of core members of the honey bee and bumblebee gut microbiota with conserved functional capabilities, confirming the common ancestry and ecology of their microbiota. However, our bacterial phylogenies were not congruent with those of the host, indicating that the evolution of the social bee gut microbiota was not driven by strict codiversification but included host switches and independent symbiont gain and losses. Finally, as reported for the honey bee and bumblebee microbiotas, we found substantial genomic divergence among strains of stingless bee gut bacteria, suggesting adaptation to different host species and glycan niches. Our study offers first insights into the genomic diversity of the stingless bee microbiota and highlights the need for broader samplings to understand the evolution of the social bee gut microbiota. IMPORTANCE Stingless bees are the most diverse group of the corbiculate bees and represent important pollinator species throughout the tropics and subtropics. They harbor specialized microbial communities in their gut that are related to those found in honey bees and bumblebees and that are likely important for bee health. Few bacteria have been cultured from the gut of stingless bees, which has prevented characterization of their genomic diversity and functional potential. Here, we established cultures of major members of the gut microbiotas of six stingless bee species and sequenced their genomes. We found that most stingless bee isolates belong to novel bacterial species distantly related to those found in honey bees and bumblebees and encoding similar functional capabilities. Our study offers a new perspective on the evolution of the social bee gut microbiota and presents a basis for characterizing the symbiotic relationships between gut bacteria and stingless bees.

Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as indicated by sCRilecs-seq.

Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the ß-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by a reduced availability of cell wall precursors at the site of cell wall synthesis due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier that is responsible for transporting these precursors across the cell membrane. The data suggest that, whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections.

Streptococcus pneumoniae is a bacterium that can cause pneumonia, meningitis and other life-threatening illnesses in humans. Currently, many S. pneumoniae infections are treated with the antibiotic amoxicillin, which kills the bacteria by weakening a structure known as the cell wall that surrounds each bacterium. However, more and more S. pneumoniae cells are becoming resistant to amoxicillin, making it harder to treat such infections. We need new ways to effectively treat S. pneumoniae infections in humans. One potential strategy would be to combine amoxicillin with another drug that boosts the activity of amoxicillin so that it is able to kill the resistant bacteria. Two drugs that both target the same process in cells are more likely to boost each other's activity. Therefore, Dewachter et al. decided to search for another drug that also weakens the cell wall of S. pneumoniae. The team first developed a new screening approach called sCRilecs-seq to silence individual genes in single S. pneumoniae cells. By looking at many cells that each had a different gene that was no longer active, the team were able to identify several genes that when silenced resulted in the cells becoming longer than normal cells (a sign the bacteria may have weak cell walls). Further experiments revealed that the cell walls of these bacteria were weaker than normal cells due to a shortage in a cell wall building material known as undecaprenyl phosphate. Dewachter et al. then demonstrated that combining an existing drug known as clomiphene ­ which is known to inhibit undecaprenyl phosphate production and is currently used to treat infertility in humans ­ together with amoxicillin is able to effectively kill S. pneumoniae that are resistant to amoxicillin alone. Clomiphene also boosted the activity of amoxicillin against S. pneumoniae that remain sensitive to the antibiotic. Before this new drug combination may be used to help treat S. pneumoniae infections in human patients, further experiments will be needed to find out the optimum dose of clomiphene to use with amoxicillin. In the future, the new screening approach developed by Dewachter et al. may also prove useful to other researchers studying a wide range of biological questions.

CRISPRi-seq for genome-wide fitness quantification in bacteria.

CRISPR interference (CRISPRi) is a powerful tool to link essential and nonessential genes to specific phenotypes and to explore their functions. Here we describe a protocol for CRISPRi screenings to assess genome-wide gene fitness in a single sequencing step (CRISPRi-seq). We demonstrate the use of the protocol in Streptococcus pneumoniae, an important human pathogen; however, the protocol can easily be adapted for use in other organisms. The protocol includes a pipeline for single-guide RNA library design, workflows for pooled CRISPRi library construction, growth assays and sequencing steps, a read analysis tool (2FAST2Q) and instructions for fitness quantification. We describe how to make an IPTG-inducible system with small libraries that are easy to handle and cost-effective and overcome bottleneck issues, which can be a problem when using similar, transposon mutagenesis-based methods. Ultimately, the procedure yields a fitness score per single-guide RNA target for any given growth condition. A genome-wide screening can be finished in 1 week with a constructed library. Data analysis and follow-up confirmation experiments can be completed in another 2-3 weeks.

Exploration of Bacterial Bottlenecks and Streptococcus pneumoniae Pathogenesis by CRISPRi-Seq.

Streptococcus pneumoniae is an opportunistic human pathogen that causes invasive diseases, including pneumonia, with greater health risks upon influenza A virus (IAV) co-infection. To facilitate pathogenesis studies in vivo, we developed an inducible CRISPR interference system that enables genome-wide fitness testing in one sequencing step (CRISPRi-seq). We applied CRISPRi-seq to assess bottlenecks and identify pneumococcal genes important in a murine pneumonia model. A critical bottleneck occurs at 48 h with few bacteria causing systemic infection. This bottleneck is not present during IAV superinfection, facilitating identification of pneumococcal pathogenesis-related genes. Top in vivo essential genes included purA, encoding adenylsuccinate synthetase, and the cps operon required for capsule production. Surprisingly, CRISPRi-seq indicated no fitness-related role for pneumolysin during superinfection. Interestingly, although metK (encoding S-adenosylmethionine synthetase) was essential in vitro, it was dispensable in vivo. This highlights advantages of CRISPRi-seq over transposon-based genetic screens, as all genes, including essential genes, can be tested for pathogenesis potential.