Systematic interrogation of CRISPR antimicrobials in Klebsiella pneumoniae reveals nuclease-, guide- and strain-dependent features influencing antimicrobial activity.

CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae as models. Comparing different Cas nucleases, DNA-targeting nucleases outperformed RNA-targeting nucleases based on the tested targets. Focusing on AsCas12a that exhibited robust targeting across different strains, we found that the elucidated modes of escape varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains, which were linked to an interplay between improper gRNA folding and strain-specific DNA repair and survival. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.

An adapted method for Cas9-mediated editing reveals the species-specific role of ß-glucoside utilization driving competition between Klebsiella species.

Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the Klebsiella oxytoca species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against Klebsiella pneumoniae. Firstly, the system allowed efficient and precise full-length gene deletion via enhanced lambda Red expression. Furthermore, we tested the efficiency of two independent, functionally validated complementation strategies. Ultimately, the insertion of universal "bookmark" targets during gene deletion subsequently allows the most optimal genetic complementation in K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. This approach offers a significant advantage by enabling the use of a single high-efficiency "bookmark" for complementing other loci or strains, eliminating the need for site-specific design. We revealed that the carbohydrate permease CasA is critical in ex vivo assays for K. pneumoniae inhibition by K. oxytoca but is neither sufficient nor required for K. michiganensis and K. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition. IMPORTANCE: Cas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in Klebsiella pneumoniae and demonstrate after optimization its utility for gene editing in three members of the Klebsiella oxytoca species complex (KoSC) namely K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. We then adapted a recently developed protocol for functional complementation based on universal "bookmark" targets applicable to all tested species. In summary, species-specific adaptation of state-of-the-art genetic tools allows efficient gene deletion and complementation in type strains as well as natural isolates of KoSC members to study microbial interactions.

Laboratory mice with a wild microbiota generate strong allergic immune responses.

Allergic disorders are caused by a combination of hereditary and environmental factors. The hygiene hypothesis postulates that early-life microbial exposures impede the development of subsequent allergic disease. Recently developed "wildling" mice are genetically identical to standard laboratory specific pathogen-free (SPF) mice but are housed under seminatural conditions and have rich microbial exposures from birth. Thus, by comparing conventional SPF mice with wildlings, we can uncouple the impact of lifelong microbial exposures from genetic factors on the allergic immune response. We found that wildlings developed larger populations of antigen-experienced T cells than conventional SPF mice, which included interleukin-10-producing CD4 T cells specific for commensal Lactobacilli strains and allergy-promoting T helper 2 (TH2) cells. In models of airway exposure to house dust mite (HDM), recombinant interleukin-33, or Alternaria alternata, wildlings developed strong allergic inflammation, characterized by eosinophil recruitment, goblet cell metaplasia, and antigen-specific immunoglobulin G1 (IgG1) and IgE responses. Wildlings developed robust de novo TH2 cell responses to incoming allergens, whereas preexisting TH2 cells could also be recruited into the allergic immune response in a cytokine-driven and TCR-independent fashion. Thus, wildling mice, which experience diverse and lifelong microbial exposures, were not protected from developing pathological allergic immune responses. Instead, wildlings mounted robust allergic responses to incoming allergens, shedding new light on the hygiene hypothesis.

Phages against Noncapsulated Klebsiella pneumoniae: Broader Host range, Slower Resistance.

Klebsiella pneumoniae (Kp), a human gut colonizer and opportunistic pathogen, is a major contributor to the global burden of antimicrobial resistance. Virulent bacteriophages represent promising agents for decolonization and therapy. However, the majority of anti-Kp phages that have been isolated thus far are highly specific to unique capsular types (anti-K phages), which is a major limitation to phage therapy prospects due to the highly polymorphic capsule of Kp. Here, we report on an original anti-Kp phage isolation strategy, using capsule-deficient Kp mutants as hosts (anti-Kd phages). We show that anti-Kd phages have a broad host range, as the majority are able to infect noncapsulated mutants of multiple genetic sublineages and O-types. Additionally, anti-Kd phages induce a lower rate of resistance emergence in vitro and provide increased killing efficiency when in combination with anti-K phages. In vivo, anti-Kd phages are able to replicate in mouse guts colonized with a capsulated Kp strain, suggesting the presence of noncapsulated Kp subpopulations. The original strategy proposed here represents a promising avenue that circumvents the Kp capsule host restriction barrier, offering promise for therapeutic development. IMPORTANCE Klebsiella pneumoniae (Kp) is an ecologically generalist bacterium as well as an opportunistic pathogen that is responsible for hospital-acquired infections and a major contributor to the global burden of antimicrobial resistance. In the last decades, limited advances have been made in the use of virulent phages as alternatives or complements to antibiotics that are used to treat Kp infections. This work demonstrates the potential value of an anti-Klebsiella phage isolation strategy that addresses the issue of the narrow host range of anti-K phages. Anti-Kd phages may be active in infection sites in which capsule expression is intermittent or repressed or in combination with anti-K phages, which often induce the loss of capsule in escape mutants.

Helicobacter spp. are prevalent in wild mice and protect from lethal Citrobacter rodentium infection in the absence of adaptive immunity.

Transfer of the gut microbiota from wild to laboratory mice alters the host's immune status and enhances resistance to infectious and metabolic diseases, but understanding of which microbes and how they promote host fitness is only emerging. Our analysis of metagenomic sequencing data reveals that Helicobacter spp. are enriched in wild compared with specific-pathogen-free (SPF) and conventionally housed mice, with multiple species commonly co-colonizing their hosts. We create laboratory mice harboring three non-SPF Helicobacter spp. to evaluate their effect on mucosal immunity and colonization resistance to the enteropathogen Citrobacter rodentium. Our experiments reveal that Helicobacter spp. interfere with C. rodentium colonization and attenuate C. rodentium-induced gut inflammation in wild-type (WT) mice, even preventing lethal infection in Rag2-/- SPF mice. Further analyses suggest that Helicobacter spp. interfere with tissue attachment of C. rodentium, putatively by reducing the availability of mucus-derived sugars. These results unveil pivotal protective functions of wild mouse microbiota constituents against intestinal infection.

Persisting Microbiota and Neuronal Imbalance Following T. gondii Infection Reliant on the Infection Route.

Toxoplasma gondii is a highly successful parasite capable of infecting all warm-blooded animals. The natural way of infection in intermediate hosts is the oral ingestion of parasite-contaminated water or food. In murine experimental models, oral infection (p.o.) of mice with T. gondii is applied to investigate mucosal and peripheral immune cell dynamics, whereas intraperitoneal infection (i.p.) is frequently used to study peripheral inflammation as well as immune cell - neuronal interaction in the central nervous system (CNS). However, the two infection routes have not yet been systematically compared along the course of infection. Here, C57BL/6 mice were infected p.o. or i.p. with a low dose of T. gondii cysts, and the acute and chronic stages of infection were compared. A more severe course of infection was detected following i.p. challenge, characterized by an increased weight loss and marked expression of proinflammatory cytokines particularly in the CNS during the chronic stage. The elevated proinflammatory cytokine expression in the ileum was more prominent after p.o. challenge that continued following the acute phase in both i.p. or p.o. infected mice. This resulted in sustained microbial dysbiosis, especially after p.o. challenge, highlighted by increased abundance of pathobionts from the phyla proteobacteria and a reduction of beneficial commensal species. Further, we revealed that in the CNS of i.p. infected mice CD4 and CD8 T cells displayed higher IFNγ production in the chronic stage. This corresponded with an increased expression of C1q and CD68 in the CNS and reduced expression of genes involved in neuronal signal transmission. Neuroinflammation-associated synaptic alterations, especially PSD-95, VGLUT, and EAAT2 expression, were more pronounced in the cortex upon i.p. infection highlighting the profound interplay between peripheral inflammation and CNS homeostasis.

Feeding a Saccharomyces cerevisiae Fermentation Product (Olimond BB) Does Not Alter the Fecal Microbiota of Thoroughbred Racehorses.

Feed supplements such as Saccharomyces cerevisiae fermentation products (SCFP) alter immune responses in horses. The purpose of this study was to analyze whether a prebiotic activity of the SCFP alters the gut microbiome in horses. Racehorses were fed either SCFP (Olimond BB, OLI, n = 6) or placebo pellets (PLA, n = 5) for 43 days. Fecal microbiota analysis was performed using 16S rRNA gene sequencing. The numbers and function of circulating immune cell subpopulations were analyzed by flow cytometry. SCFP supplementation resulted in non-consistent differences in fecal microbiota between the PLA and OLI during the feeding period. Rather, the individual animal had the highest impact on fecal microbiota composition. OLI and PLA horses displayed the same changes in numbers of blood leukocyte subpopulations over time. One day after a booster vaccination against equine influenza during the feeding period, the alpha diversity of fecal microbiota of PLA horses was significantly higher compared to OLI horses. This suggests that SCFP feeding altered the vaccination-induced spectrum of released mediators, potentially affecting gut microbiota. The overall non-consistent findings argue against a strong prebiotic effect of Olimond BB on the microbiota in racehorses. Fecal microbiota differences between the groups were also noticed outside the feeding period and, hence, are most likely not caused by the SCFP additive.

Klebsiella oxytoca causes colonization resistance against multidrug-resistant K. pneumoniae in the gut via cooperative carbohydrate competition.

Gut colonization with multidrug-resistant (MDR) bacteria enhances the risk of bloodstream infections in susceptible individuals. We demonstrate highly variable degrees of ex vivo colonization resistance against a carbapenem-resistant Klebsiella pneumoniae strain in human feces samples and subsequently isolate diverse K. oxytoca strains from protected donors. Several of these K. oxytoca strains reduce gut colonization of MDR K. pneumoniae strains in antibiotic-treated and gnotobiotic mouse models. Comparative analysis of K. oxytoca strains coupled with CRISPR-Cas9-mediated deletion of casA, a protein essential for utilization of selected beta-glucosides, identified competition for specific carbohydrates as key in promoting colonization resistance. In addition to direct competition between K. oxytoca and K. pneumoniae, cooperation with additional commensals is required to reestablish full colonization resistance and gut decolonization. Finally, humanized microbiota mice generated from K. pneumoniae-susceptible donors are protected by K. oxytoca administration, demonstrating the potential of commensal K. oxytoca strains as next-generation probiotics.

Curbing gastrointestinal infections by defensin fragment modifications without harming commensal microbiota.

The occurrence and spread of multidrug-resistant pathogens, especially bacteria from the ESKAPE panel, increases the risk to succumb to untreatable infections. We developed a novel antimicrobial peptide, Pam-3, with antibacterial and antibiofilm properties to counter this threat. The peptide is based on an eight-amino acid carboxyl-terminal fragment of human ß-defensin 1. Pam-3 exhibited prominent antimicrobial activity against multidrug-resistant ESKAPE pathogens and additionally eradicated already established biofilms in vitro, primarily by disrupting membrane integrity of its target cell. Importantly, prolonged exposure did not result in drug-resistance to Pam-3. In mouse models, Pam-3 selectively reduced acute intestinal Salmonella and established Citrobacter infections, without compromising the core microbiota, hence displaying an added benefit to traditional broad-spectrum antibiotics. In conclusion, our data support the development of defensin-derived antimicrobial agents as a novel approach to fight multidrug-resistant bacteria, where Pam-3 appears as a particularly promising microbiota-preserving candidate.

Variations in microbiota composition of laboratory mice influence Citrobacter rodentium infection via variable short-chain fatty acid production.

The composition of the intestinal microbiota influences the outcome of enteric infections in human and mice. However, the role of specific members and their metabolites contributing to disease severity is largely unknown. Using isogenic mouse lines harboring distinct microbiota communities, we observed highly variable disease kinetics of enteric Citrobacter rodentium colonization after infection. Transfer of communities from susceptible and resistant mice into germ-free mice verified that the varying susceptibilities are determined by microbiota composition. The strongest differences in colonization were observed in the cecum and could be maintained in vitro by coculturing cecal bacteria with C. rodentium. Cohousing of animals as well as the transfer of cultivable bacteria from resistant to susceptible mice led to variable outcomes in the recipient mice. Microbiome analysis revealed that a higher abundance of butyrate-producing bacteria was associated with the resistant phenotype. Quantification of short-chain fatty acid (SCFA) levels before and after infection revealed increased concentrations of acetate, butyrate and propionate in mice with delayed colonization. Addition of physiological concentrations of butyrate, but not of acetate and/or propionate strongly impaired growth of C. rodentium in vitro. In vivo supplementation of susceptible, antibiotic-treated and germ-free mice with butyrate led to the same level of protection, notably only when cecal butyrate concentration reached a concentration higher than 50 nmol/mg indicating a critical threshold for protection. In the recent years, commensal-derived primary and secondary bacterial metabolites emerged as potent modulators of hosts susceptibility to infection. Our results provide evidence that variations in SCFA production in mice fed fibre-rich chow-based diets modulate susceptibility to colonization with Enterobacteriaceae not only in antibiotic-disturbed ecosystems but even in undisturbed microbial communities. These findings emphasise the need for microbiota normalization across laboratory mouse lines for infection experiments with the model-pathogen C. rodentium independent of investigations of diet and antibiotic usage.

c-FLIP is crucial for IL-7/IL-15-dependent NKp46+ ILC development and protection from intestinal inflammation in mice.

NKp46+ innate lymphoid cells (ILC) modulate tissue homeostasis and anti-microbial immune responses. ILC development and function are regulated by cytokines such as Interleukin (IL)-7 and IL-15. However, the ILC-intrinsic pathways translating cytokine signals into developmental programs are largely unknown. Here we show that the anti-apoptotic molecule cellular FLICE-like inhibitory protein (c-FLIP) is crucial for the generation of IL-7/IL-15-dependent NKp46+ ILC1, including conventional natural killer (cNK) cells, and ILC3. Cytokine-induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) precedes up-regulation of c-FLIP, which protects developing NKp46+ ILC from TNF-induced apoptosis. NKp46+ ILC-specific inactivation of c-FLIP leads to the loss of all IL-7/IL-15-dependent NKp46+ ILC, thereby inducing early-onset chronic colitis and subsequently microbial dysbiosis; meanwhile, the depletion of cNK, but not NKp46+ ILC1/3, aggravates experimental colitis. In summary, our data demonstrate a non-redundant function of c-FLIP for the generation of NKp46+ ILC, which protect T/B lymphocyte-sufficient mice from intestinal inflammation.

The interferon-stimulated gene product oligoadenylate synthetase-like protein enhances replication of Kaposi's sarcoma-associated herpesvirus (KSHV) and interacts with the KSHV ORF20 protein.

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few oncogenic human viruses known to date. Its large genome encodes more than 85 proteins and includes both unique viral proteins as well as proteins conserved amongst herpesviruses. KSHV ORF20 is a member of the herpesviral core UL24 family, but the function of ORF20 and its role in the viral life cycle is not well understood. ORF20 encodes three largely uncharacterized isoforms, which we found were localized predominantly in the nuclei and nucleoli. Quantitative affinity purification coupled to mass spectrometry (q-AP-MS) identified numerous specific interacting partners of ORF20, including ribosomal proteins and the interferon-stimulated gene product (ISG) oligoadenylate synthetase-like protein (OASL). Both endogenous and transiently transfected OASL co-immunoprecipitated with ORF20, and this interaction was conserved among all ORF20 isoforms and multiple ORF20 homologs of the UL24 family in other herpesviruses. Characterization of OASL interacting partners by q-AP-MS identified a very similar interactome to that of ORF20. Both ORF20 and OASL copurified with 40S and 60S ribosomal subunits, and when they were co-expressed, they associated with polysomes. Although ORF20 did not have a global effect on translation, ORF20 enhanced RIG-I induced expression of endogenous OASL in an IRF3-dependent but IFNAR-independent manner. OASL has been characterized as an ISG with antiviral activity against some viruses, but its role for gammaherpesviruses was unknown. We show that OASL and ORF20 mRNA expression were induced early after reactivation of latently infected HuARLT-rKSHV.219 cells. Intriguingly, we found that OASL enhanced infection of KSHV. During infection with a KSHV ORF20stop mutant, however, OASL-dependent enhancement of infectivity was lost. Our data have characterized the interaction of ORF20 with OASL and suggest ORF20 usurps the function of OASL to benefit KSHV infection.