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Malaria is a highly lethal infectious disease caused by Plasmodium parasites. These parasites are transmitted to vertebrate hosts when mosquitoes of the Anopheles genus probe for a blood meal. Sporozoites, the infectious stage of Plasmodium, transit to the liver within hours of injection into the dermis. Vaccine efforts are hindered by the complexity of the parasite's lifecycle and the speed at which the infection is established in the liver. In an effort to enhance immunity against Plasmodium, we produced a virus-like particle (VLP)-based vaccine displaying an epitope of TRIO, an Anopheles salivary protein that has been shown to enhance mobility and dispersal of sporozoites in the dermis. Previous work demonstrated that passive immunization with TRIO offered protection from liver infection and acted synergistically with a Plasmodium-targeted vaccine. Immunization of mice with TRIO VLPs resulted in high-titer and long-lasting antibody responses that did not significantly drop for over 18 months post-immunization. TRIO VLPs were similarly immunogenic when combined with an anti-malaria vaccine targeting the L9 epitope of the Plasmodium falciparum circumsporozoite protein. However, when used in a malaria challenge mouse model, TRIO VLPs only provided modest protection from infection and did not boost the protection provided by L9 VLPs.IMPORTANCEProteins present in the salivary glands of mosquitos have been shown to enhance the transmission efficiency of mosquito-borne pathogens, suggesting that interventions targeting the activity of these proteins could reduce transmission. Here, we looked at the efficacy of a vaccine targeting TRIO, an Anopheles mosquito salivary protein that has been reported to enhance Plasmodium falciparum malaria infection. We show that this vaccine can elicit strong anti-TRIO antibody responses, but these antibodies only result in a modest decrease in infection.

Gina Lewin works in the field of microbial ecology, with a focus on the human microbiota. In this mSphere of Influence article, she reflects on how two papers describing bacterial single-cell RNA-seq-"Prokaryotic single-cell RNA sequencing by in situ combinatorial indexing" by S. B. Blattman, W. Jiang, P. Oikonomou, and S. Tavazoie (Nat Microbiol 5:1192-1201, 2020, https://doi.org/10.1038/s41564-020-0729-6) and "Microbial single-cell RNA sequencing by split-pool barcoding" by A. Kuchina, L. M. Brettner, L. Paleologu, C. M. Roco, et al. (Science 371:eaba5257, 2021, https://doi.org/10.1126/science.aba5257)-impacted her work by developing a new approach to study how single cells of bacteria contribute to ecosystem-level processes.

We previously reported that mutations outside the spike protein play a role in the attenuation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.1 variant in human ACE2 transgenic mice (K18-hACE2). Here, we assessed the pathogenicity of SARS-CoV-2 (WA1/2020) containing mutations from the Omicron BA.1 variant in K18-hACE2 mice. At an infection dose of 10⁴ plaque-forming units (PFU), WA1 virus carrying Omicron BA.1 Nsp14(I42V), E(T9I), M(D3G/Q19E/A63T), but not Nsp6(Δ105-107, I189V), substitutions showed significant reduction in lethality. Interestingly, reduction of viral load is more pronounced in the brains than in the lungs. Subsequent analyses suggest that BA.1 E(T9I) and M(D3G/Q19E/A63T) substitutions result in less efficient packaging of virus-like particles. Given that Nsp14(I42V), E(T9I), M(Q19E/A63T) are well preserved in subsequent omicron subvariants, including currently circulating variants, our findings highlight the importance of understanding how non-spike mutations affect the pathogenicity of SARS-CoV-2 variants.

Importance: Inoculation of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) with SARS-CoV-2 often leads to a fatal brain infection. Omicron BA.1 variant, however, was found to be non-lethal in this model. Here, we systematically assessed the effect of individual mutations of Omicron BA.1 on the pathogenicity of the virus in hACE2 transgenic mice and found that combination of 5 mutations of Nsp14, E, and M of BA.1 variant significantly lowered brain viral load and reduced lethality. These results provide new insights into how SARS-CoV-2 Omicron BA.1 is attenuated.

Visceral leishmaniasis (VL) is a vector-borne disease caused by the obligate intracellular protozoan Leishmania donovani in India. VL can be complicated by post-kala-azar dermal leishmaniasis (PKDL), a macular or nodular rash that develops in 10%-20% of patients after treatment of VL in India. Patients with PKDL are infectious to sand flies, promoting further transmission of the parasite. MicroRNAs (miRNAs) are 18-25 nt, non-coding RNAs that simultaneously regulate the expression of several or many target transcripts. This study was based on the hypothesis that the host response to L. donovani is modified by distinct sets of miRNAs in VL or PKDL and that these might differ from healthy controls. We investigated this hypothesis using a NanoString panel to profile the miRNAs expressed in the plasma of patients with VL or PKDL diagnosed at a hospital in Bihar, India. We compared these to plasma microRNAs of healthy control individuals from the same endemic villages. miRNAs hsa-miR-223-3p, hsa-miR-191-5p, hsa-miR-23a-3p, and hsa-1285-5p were significantly higher in the plasma samples from patients with VL compared to either PKDL or endemic controls. Prediction programs highlighted potential mRNA targeted by these miRNAs, among which we verified the down-modulation of several transcripts belonging to the NFκB and NLRP3 inflammasome pathways in circulating leukocytes of VL patients. By contrast, miRNA patterns in subjects with PKDL were similar to control subjects, possibly suggesting that the pathogenic immune response during PKDL is primarily localized in the skin.IMPORTANCEInfection of humans with the protozoan Leishmania donovani can be asymptomatic or it can cause fatal visceral leishmaniasis (VL), sometimes followed by the cutaneous complication PKDL. Parasites are spread through sand fly bites in endemic regions, and parasites in post-kala-azar dermal leishmaniasis (PKDL) skin lesions are a source of prolonged parasite transmission to sand flies, compromising disease eradication efforts. Since microRNAs can simultaneously modify the expression of multiple genes, we examined microRNAs in the blood that might be partial determinants of pathogenic responses leading to VL or PKDL. Our studies revealed several miRNAs expressed that are elevated in the plasma of patients with VL, which suppress some of the inflammatory responses that promote parasite killing. However, miRNA profiles were very similar between PKDL patients and controls, raising the possibility that major factors that lead to prolonged retention of parasites in the skin during PKDL are not systemic but are localized in the skin.

Porphyromonas gingivalis (P. gingivalis), a gram-negative, black-pigmented anaerobe, is a major etiological agent and a leading cause of periodontitis. Fimbriae protein Mfa1 is a key virulence factor of P. gingivalis and plays a crucial role in bacterial adhesion, colonization, biofilm formation, and persistent inflammation, making it a promising therapeutic target. However, the role of anti-Mfa1 antibodies and the underlying protective mechanisms remain largely unexplored. Here, we developed and characterized the monoclonal antibodies (mAbs) targeting the Mfa1 protein of P. gingivalis. Function analysis showed that anti-Mfa1 mAbs mediated bacterial agglutination and inhibited P. gingivalis adhesion to saliva-coated hydroxyapatite and host cells. Notably, anti-Mfa1 mAbs significantly reduced bacterial burden and alveolar bone loss in a P. gingivalis-induced experimental periodontitis model. These results show that anti-Mfa1 mAbs can be beneficial in alleviating P. gingivalis infections, and provide important insights for the development of adequate adjuvant treatment regimens for Mfa1-targeted therapeutics.

Importance: Fimbriae (pili) play an important role in bacterial adhesion, invasion of host cells and tissues, and formation of biofilms. Studies have shown that two types of fimbriae of Porphyromonas gingivalis, FimA and Mfa1, are important for colonization and infection through their binding to host tissues and other bacteria. While anti-FimA antibodies have been shown to improve periodontitis, the effect of anti-Mfa1 antibodies on P. gingivalis infection and periodontitis was previously unknown. In this study, we report for the first time that anti-Mfa1 monoclonal antibodies can reduce P. gingivalis infection and improve periodontitis. These findings suggest that Mfa1 represents a promising therapeutic target, and the development of anti-Mfa1 mAbs holds a potential as essential diagnostic and adjunctive therapeutic tools for managing P. gingivalis-related diseases.

The eukaryotic CCR4-NOT deadenylase complex is a highly conserved regulator of mRNA metabolism that influences the expression of the complete transcriptome, representing a prime target for a generalist bacterial pathogen. We show that a translocated bacterial effector protein, PieF (Lpg1972) of Legionella pneumophila, directly interacts with the CNOT7/8 nuclease module of CCR4-NOT, with a dissociation constant in the low nanomolar range. PieF is a robust in vitro inhibitor of the DEDD-type nuclease, CNOT7, acting in a stoichiometric, dose-dependent manner. Heterologous expression of PieF phenocopies knockout of the CNOT7 ortholog (POP2) in Saccharomyces cerevisiae, resulting in 6-azauracil sensitivity. In mammalian cells, expression of PieF leads to a variety of quantifiable phenotypes: PieF silences gene expression and reduces mRNA steady-state levels when artificially tethered to a reporter transcript, and its overexpression results in the nuclear exclusion of CNOT7. PieF expression also disrupts the association between CNOT6/6L EEP-type nucleases and CNOT7. Adding to the complexities of PieF activity in vivo, we identified a separate domain of PieF responsible for binding to eukaryotic kinases. Unlike what we observe for CNOT6/6L, we show that these interactions can occur concomitantly with PieF's binding to CNOT7. Collectively, this work reveals a new, highly conserved target of L. pneumophila effectors and suggests a mechanism by which the pathogen may be modulating host mRNA stability and expression during infection.

Importance: The intracellular bacterial pathogen Legionella pneumophila targets conserved eukaryotic pathways to establish a replicative niche inside host cells. With a host range that spans billions of years of evolution (from protists to humans), the interaction between L. pneumophila and its hosts frequently involves conserved eukaryotic pathways (protein translation, ubiquitination, membrane trafficking, autophagy, and the cytoskeleton). Here, we present the identification of a new, highly conserved host target of L. pneumophila effectors: the CCR4-NOT complex. CCR4-NOT modulates mRNA stability in eukaryotes from yeast to humans, making it an attractive target for a generalist pathogen, such as L. pneumophila. We show that the uncharacterized L. pneumophila effector PieF specifically targets one component of this complex, the deadenylase subunit CNOT7/8. We show that the interaction between PieF and CNOT7 is direct, occurs with high affinity, and reshapes the catalytic activity, localization, and composition of the complex across evolutionarily diverse eukaryotic cells.

Advances in technology have facilitated extensive sample collection to explore microbiomes across diverse systems, leading to a growing reliance on ultracold freezers for storing both samples and extracted DNA. However, freezer malfunctions can jeopardize data integrity. To evaluate the impact of an unexpected -80°C freezer failure and the recoverability of thawed soil samples, we extracted DNA and compared it to long-term DNA stored at -20°C and original 16S and ITS2 sequencing data collected before the malfunction. Using Illumina MiSeq, we assessed how the freezer failure and long-term storage influenced the resilience of bacterial or fungal richness or community composition and our ability to accurately determine experimental treatment effects. Our results reveal substantial resilience in fungal richness and both bacterial and fungal beta-diversity to soil sample thawing and extended frozen DNA storage. This resilience facilitated biological inferences that closely mirrored those observed in the original study. Notably, fungi exhibited greater resilience to short-term thawing compared to bacteria, which showed sensitivity to both thawing and long-term freezing. Moreover, taxonomic composition analysis revealed the persistence of dominant microbial taxa under thawing and prolonged freezing, suggesting that dominant microbes remain viable for tracking across temporal studies. In conclusion, our study highlights that beta-diversity is more robust than alpha-diversity and fungi are more resilient to freezer failure than bacteria. Furthermore, our findings underscore the effectiveness of soil storage at -80°C compared to storage of extracted DNA at -20°C, despite potential freezer failures, as the most robust method for long-term storage in microbiome studies.

Importance: Microbiome studies heavily rely on ultracold freezers for sample storage. Unfortunately, these freezers are prone to frequent malfunctions, resulting in the loss of invaluable samples at laboratories worldwide. Such losses can halt research progress due to potential issues with sample reliability. Our research demonstrates that not all is lost when an unforeseen freezer failure occurs. Samples can still be reliably used to assess treatment effects, which is particularly important for long-term temporal studies where samples cannot be readily obtained again.

Post-transcriptional gene regulation by non-coding small RNAs (sRNAs) is critical for colonization and survival of enteric pathogens, including the zoonotic pathogen Campylobacter jejuni. In this study, we utilized C. jejuni IA3902 (a representative isolate of the sheep abortion clone) and C. jejuni W7 (a highly motile variant of NCTC 11168, a human gastroenteritis strain) to further investigate regulation by sRNA CjNC110. Both motility and autoagglutination ability were confirmed to be phenotypes of conserved regulation by CjNC110. However, we demonstrated that W7∆CjNC110 does not change chicken colonization levels compared to W7 wild type, directly contrasting IA3902∆CjNC110, which had decreased colonization ability. Subsequently, we determined strain-specific phenotype variation between W7∆CjNC110 and IA3902∆CjNC110 when examining intracellular L-methionine (L-met) levels controlled by the activated methyl cycle (AMC). We hypothesized that the presence of a secondary system for L-met production conferred by MetAB in W7 but not IA3902 might explain the difference in both chicken colonization and L-met availability. Insertion of metAB within IA3902∆CjNC110 (naturally absent) restored intracellular L-met levels in IA3902∆CjNC110::metAB and overcame the colonization defect that resulted from mutagenesis of CjNC110 in IA3902. Deletion of metAB in W7∆CjNC110 (naturally present) led to a decrease in L-met in W7∆CjNC110∆metAB and a colonization defect which was otherwise masked in W7∆CjNC110. Our results indicate that regulation of the AMC leading to altered L-met availability is a conserved regulatory function of CjNC110 in C. jejuni and confirm that L-met generation via the AMC as activated by CjNC110 is critical for optimal host colonization.IMPORTANCEDuring this study, the regulatory action and conservation of function of CjNC110 between two different zoonotically important Campylobacter jejuni strains were examined. Critically, this work for the first time reveals regulation of L-methionine (L-met) production within the activated methyl cycle (AMC) by small RNA (sRNA) CjNC110 as a key factor driving C. jejuni optimal chicken colonization. As a growing body of evidence suggests that maintenance of L-met homeostasis appears to be critical for C. jejuni colonization, interventions targeting the AMC could provide a critical control point for therapeutic drug options to combat this zoonotic pathogen. Our results also indicate that even for conserved sRNAs such as CjNC110, strain-specific differences in phenotypes regulated by sRNAs may exist, independent of conserved regulatory action. Depending on the strain examined and accessory genomic content present, conserved regulatory actions might be masked, thus investigation in multiple strains may be warranted.

Nucleic acid amplification testing (NAAT) for N. gonorrhoeae is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting N. gonorrhoeae. We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical urine specimens, and optimize assay kinetics. We collected urine specimens among men presenting with urethritis enrolling in a clinical trial at the Massachusetts General Hospital Sexual Health Clinic. We assessed the quantified DNA yield of detergent-based extractions with and without heat. We selected one detergent for extracting all specimens, paired with isothermal recombinase polymerase amplification for 90 minutes and lateral flow Cas13a detection, interpreted via pixel intensity analysis. We also trained a smartphone-based machine-learning model on 1,008 images to classify lateral flow results. We used the model to interpret lateral flow results from the clinical specimens. We also tested a modified amplification chemistry with a second forward primer lacking the T7-promoter to accelerate reaction kinetics. Extraction with 0.02% Triton X resulted in an average DNA yield of 2.6 × 10⁶ copies/µL (SD ± 6.7 × 10⁵). We treated 40 urine specimens (n = 12 positive) with 0.02% Triton X, and using quantified pixel intensity analysis, the Cas13a-based assay correctly classified all specimens (100% agreement; 95% CI 91.2%-100%). The machine-learning model correctly classified 45/45 strips in the validation data set and all 40 lateral flow strips from clinical specimens. Including the second forward primer reduced incubation time to 60 minutes. Using point-of-care DNA extraction, our Cas13a-based lateral flow N. gonorrhoeae assay demonstrated promising performance among clinical urine specimens.IMPORTANCEUsing a CRISPR-based assay we previously developed for Neisseria gonorrhoeae detection, we developed new techniques to facilitate point-of-care use. We then demonstrated the promising performance of that assay in clinical specimens. Furthermore, we developed a smartphone-based machine learning application for assisting interpretation of lateral flow strip results. Such an assay has the potential to transform the care of sexually transmitted infections in low-resource settings where diagnostic tests are unavailable. A point-of-care pathogen-specific assay, paired with the connectivity offered by a smartphone application, can also support public health surveillance efforts in such areas.