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In March 2024, highly pathogenic H5N1 was detected in dairy cows; as of 12 December 2024, it had spread to over 800 herds in 16 states. The ongoing outbreak is a public health crisis affecting both humans and animals, as interspecies transmission has emerged as a common characteristic of this virus. As of 12 December 2024, >30 humans have been infected in the United States related to dairy cow exposure. In this mGem, we discuss transmission modalities between cows within herds, the spread of the virus between dairy farms, and exposure risks for humans. We also highlight major gaps in knowledge constituting barriers to our ability to effectively control the spread of H5N1 in dairy cows and reduce the risks to humans.

White spot syndrome virus (WSSV) is a debilitating viral pathogen that poses a significant threat to the global crustacean farming industry. It has a wide host tropism because it uses several receptors to facilitate its attachment and entry. Thus far, not all the receptors have been identified. Here, we employed a BioID-based screening method to identify the Na⁺-K⁺-ATPase alpha subunit (PvATP1A) as a potential receptor in Penaeus vannamei. Although during the early stages of WSSV infection, PvATP1A was induced and underwent oligomerization, clustering, and internalization, knockdown of PvATP1A inhibited viral entry and replication. PvATP1A interacted with the WSSV envelope protein VP28 through its multiple extracellular regions, whereas synthetic PvATP1A extracellular region peptides blocked WSSV entry and replication. We showed that PvATP1A did not affect WSSV attachment but facilitated internalization via caveolin-mediated endocytosis and macropinocytosis. These findings provide a robust receptor screening approach that identified PvATP1A as an entry receptor for WSSV, presenting a novel target for the development of anti-WSSV therapeutics.

Importance: Cell surface receptors are crucial for mediating virus entry into host cells. Identification and characterization of virus receptors are fundamental yet challenging aspects of virology research. In this study, a BioID-based screening method was employed to identify the Na⁺-K⁺-ATPase alpha subunit (PvATP1A) as a potential receptor for white spot syndrome virus (WSSV) in the shrimp Penaeus vannamei. We demonstrated that PvATP1A interacted with the WSSV envelope protein VP28 via its multiple extracellular regions, thereby promoting viral internalization through caveolin-mediated endocytosis and macropinocytosis. Importantly, compared with previously identified WSSV receptors such as β-integrin, glucose transporter 1 (Glut1), and polymeric immunoglobulin receptor (pIgR), PvATP1A demonstrated significantly enhanced viral entry, indicating that PvATP1A is a crucial entry receptor of WSSV. This study not only presents a robust approach for screening virus receptors but also identifies PvATP1A as a promising target for the development of anti-WSSV therapeutics.

The systemic spread of viruses in plants requires successful viral cell-to-cell movement through plasmodesmata (PD). Viral movement proteins (MPs) interact with cellular proteins to modify and utilize host transport routes. Broad bean wilt virus 2 (BBWV2) moves from cell to cell as a virion through the PD gated by VP37, the MP of BBWV2. However, the host proteins that function in the cell-to-cell movement of BBWV2 remain unclear. In this study, we identified cellular heat shock protein 90 (HSP90) as an interacting partner of VP37. The interaction between HSP90 and VP37 was assessed using the yeast two-hybrid assay, co-immunoprecipitation, and bimolecular fluorescence complementation. Tobacco rattle virus-based virus-induced gene silencing analysis revealed that HSP90 silencing significantly inhibited the systemic spread of BBWV2 in Nicotiana benthamiana plants. Furthermore, in planta treatment with geldanamycin (GDA), an inhibitor of the chaperone function of HSP90, demonstrated the necessity of HSP90 in successful cell-to-cell movement and systemic infection of BBWV2. Interestingly, GDA treatment inhibited the HSP90-VP37 interaction at the PD, resulting in the inhibition of VP37-derived tubule formation through the PD. Our results suggest that the HSP90-VP37 interaction regulates VP37-derived tubule formation through the PD, thereby facilitating the cell-to-cell movement of BBWV2.IMPORTANCEThis study highlights the regulatory role of heat shock protein 90 (HSP90) in facilitating the cell-to-cell movement of broad bean wilt virus 2 (BBWV2). HSP90 interacted with VP37, the movement protein of BBWV2, specifically at plasmodesmata (PD). This study demonstrated that the HSP90-VP37 interaction is crucial for viral cell-to-cell movement and the formation of VP37-derived tubules, which are essential structures for virus transport through the PD. The ATP-dependent chaperone activity of HSP90 is integral to this interaction, as demonstrated by the inhibition of virus movement upon treatment with geldanamycin, which disrupts the function of HSP90. These findings elucidate the molecular mechanisms underlying the cell-to-cell movement of plant viruses and highlight the role of HSP90 in viral infection. This study suggests that the chaperone activity of HSP90 may function in changing the conformational structure of VP37, thereby facilitating the assembly and function of virus-induced structures required for viral cell-to-cell movement.

Peptidoglycan (PG)-modifying enzymes play a crucial role in cell wall remodeling, essential for growth and division. Cell wall degradation products are transported to the cytoplasm and recycled back in most gram-negative bacteria, and PG recycling is also linked to β-lactam resistance in many bacteria. Caulobacter crescentus is intrinsically resistant to β-lactams. Recently, it was shown that a soluble lytic transglycosylase, SdpA, is essential for β-lactam resistance. However, the precise role of SdpA in β-lactam resistance is unknown. This study investigated the PG recycling pathway and its role in antibiotic resistance in C. crescentus. Anhydromuropeptides generated by the action of lytic transglycosylases (LTs) are transported to the cytoplasm by the permease AmpG. C. crescentus encodes an ampG homolog, and deletion mutants of sdpA and ampG are sensitive to β-lactams. The ampG deletion mutant displays a significant accumulation of anhydromuropeptides in the periplasm of C. crescentus, demonstrating its essential role in PG recycling. While single knockout mutants of sdpA and ampG exhibit no growth defects, double-deletion mutants (∆sdpA∆ampG) exhibit severe growth and morphological defects. These double mutants also show enhanced sensitivity to β-lactams. Analysis of soluble muropeptides in wild-type (WT), ∆sdpA, and ∆ampG mutants revealed reduced levels of PG precursors (UDP-GlcNAc, UDP-MurNAc, and UDP-MurNAc-P5), suggesting that PG recycling products contribute toward de novo PG biosynthesis. Furthermore, supplementing the growth media with GlcNAc sugar enhanced the fitness of ∆sdpA and ∆ampG mutants under β-lactam stress. In conclusion, our study indicates that defects in PG recycling compromise cell wall biogenesis, leading to antibiotic sensitivity in C. crescentus.IMPORTANCEβ-lactam antibiotics target the peptidoglycan cell wall biosynthetic pathway in bacteria. In response to antibiotic pressures, bacteria have developed various resistance mechanisms. In many gram-negative species, cell wall degradation products are transported into the cytoplasm and induce the expression of β-lactamase enzymes. In this study, we investigated the cell wall recycling pathway and its role in antibiotic resistance in Caulobacter crescentus. Based on our data and prior studies, we propose that cell wall degradation products are utilized for the synthesis of peptidoglycan precursors in the cytoplasm. A deficiency in cell wall recycling leads to cell wall defects and increased antibiotic sensitivity in C. crescentus. These findings are crucial for understanding antibiotic resistance mechanisms in bacteria.

The bacterium Listeria monocytogenes (Lm) causes listeriosis in humans and ruminants. Acute lesions are predominantly infiltrated by polymorphonuclear neutrophils (PMNs), considered to be the efficient bactericidal arm of innate immunity. However, recent evidence suggests that PMNs cannot achieve antilisterial sterilizing immunity and that Lm may persist within PMNs. Despite this, interactions between PMNs and Lm remain poorly understood. In this study, we characterized the listericidal activity and interaction dynamics of bovine PMNs with Lm ex vivo. Phagocytosed Lm failed to escape into the PMN cytosol and was primarily targeted by phagolysosomal mechanisms. However, PMNs enabled prolonged intravacuolar survival of a resilient Lm subpopulation, largely as viable but non-culturable (VBNC) bacteria. This resilient Lm population could spread from PMNs to a cell line, resuscitate, and complete its canonical life cycle, thereby perpetuating the infection. Therefore, we identify PMNs as a mobile niche for Lm survival and provide evidence that PMNs harbor VBNC bacteria, potentially facilitating Lm dissemination within the host.

Importance: Listeria monocytogenes (Lm) is a significant foodborne pathogen responsible for high hospitalization rates in humans, especially vulnerable groups such as the elderly, pregnant women, and immunocompromised individuals. In animals like ruminants, Lm infection leads to severe disease manifestations, notably brainstem encephalitis. This study uncovers a novel mechanism by which bovine neutrophils (PMNs) harbor Lm in a viable but non-culturable (VBNC) state, enabling the bacteria to hide in the host. PMNs, traditionally viewed as bacteria killers, may serve as Trojan horses, allowing Lm to persist and spread within the host. This discovery has broad implications for understanding Lm's persistence, its role in recurrent infections, and the development of new therapeutic strategies targeting VBNC forms of Lm to improve treatment outcomes and disease control.

Fusobacterium nucleatum (Fn) is an oral commensal inhabiting the human gingival plaque that is rarely found in the gut. However, in colorectal cancer (CRC), Fn can be isolated from stool samples and detected in metagenomes. We hypothesized that ecological characteristics of the gut are altered by disease, enabling Fn to colonize. Multiple genomically distinct populations of Fn exist, but their ecological preferences are unstudied. We identified six well-separated populations in 133 Fn genomes and used simulated metagenomes to demonstrate sensitive detection of populations in human oral and gut metagenomes. In 9,560 samples from 11 studies, Fn population C2 animalis is elevated in gut metagenomes from CRC and Crohn's disease patients and is observed more frequently in CRC stool samples than in the gingiva. Polymorphum, the most prevalent gingival Fn population, is significantly increased in Crohn's stool samples; this effect was significantly stronger in male hosts than in female. We find polymorphum genomes are enriched for biosynthetic gene clusters and fluoride exporters, while C2 animalis are high in iron transporters. Fn populations thus associate with specific clinical and demographic phenotypes and harbor distinct functional features. Ecological differences in closely related groups of bacteria inform microbiome impacts on human health.

Importance: Fusobacterium nucleatum is a bacterium normally found in the gingiva. F. nucleatum generally does not colonize the healthy gut, but is observed in approximately a third of colorectal cancer (CRC) patient guts. F. nucleatum's presence in the gut during CRC has been linked to worse prognosis and increased tumor proliferation. Here, we describe the population structure of F. nucleatum in oral and gut microbiomes. We report substantial diversity in gene carriage among six distinct populations of F. nucleatum and identify population disease and body-site preferences. We find the C2 animalis population is more common in the CRC gut than in the gingiva and is enriched for iron transporters, which support gut colonization in known pathogens. We find that C2 animalis is also enriched in Crohn's disease and type 2 diabetes, suggesting ecological commonalities between the three diseases. Our work shows that closely related bacteria can have different associations with human physiology.

T follicular helper (Tfh) cells are crucial for B cell activation and subsequent antibody production. This functionality is influenced by surface markers such as CD40L, a costimulatory factor which promotes B cell activation, and CD57, which is a well-known marker of senescence. This study examined age-specific differences in Tfh cell function in Bangladeshi and American children. At age two, Bangladeshi children displayed impaired CD40L upregulation and significant CD57 downregulation upon stimulation. These patterns, not observed in American children of the same age, suggested an exhaustion-like phenotype potentially driven by environmental factors. Random forest and generalized estimating equations (GEE) modeling was used to analyze predictors of Tfh cell response to stimulation. Days since the last antibiotic treatment, total antibiotic treatments, diarrheal episodes, and malnutrition were identified as variables that significantly impacted the Tfh response to stimuli. To assess Tfh cell ability to promote antibody responses, we correlated Tfh functionality with antibody concentration post-vaccination and in response to infection with Cryptosporidium, an endemic apicomplexan parasite. Increased CD40L expression upon stimulation correlated positively with anti-Poliovirus type 2/3 neutralizing antibody and anti-Cp17 (a Cryptosporidium sporozoite antigen) IgA concentrations. In contrast, increased CD57 expression was significantly correlated with decreased anti-Cp17 IgA. This indicates that an activation-supportive phenotype (CD40L+) may be more effective in promoting immunity than a senescent phenotype (CD57+). Together, these findings suggest that early-life environmental exposures may program Tfh cell functionality, impacting immune response potential in settings with high pathogen exposure.

Importance: T follicular helper (Tfh) cells are upstream mediators that shape the humoral immune response to specific antigens. The generation of an effective memory response to infection is vital to prevent subsequent reinfections. However, in areas with high burdens of exposure to infections, such as the urban community from Bangladesh studied here, children are consistently exposed to inflammatory pathogens. Specific environmental exposures significantly influenced Tfh cell activation and senescence phenotypes. Additionally, Tfh cell responses correlated with antibody concentrations following vaccination or infection, indicating that environmental factors may play a critical role in shaping effective immunity in early childhood.

Type II toxin-antitoxin (TA) systems are widespread in prokaryotes. They consist of neighboring genes encoding two small proteins: a toxin that inhibits a critical cellular process and an antitoxin that binds to and neutralizes the toxin. The VapD nuclease and the VapX antitoxin comprise a type II TA system that contributes to the virulence of the human pathogen Haemophilus influenzae. We analyzed the diversity and evolution of VapD-like proteins. By examining loci adjacent to genes coding for VapD-like proteins, we identified two novel families of antitoxins, which we named VapY and VapW. VapD toxins cognate to novel antitoxins induce the SOS response when overproduced, suggesting they target cellular processes related to genomic DNA integrity, maintenance, or replication. Though VapY has no sequence similarity to VapX, they share the same SH3 fold characterized by the five anti-parallel β sheets that form a barrel. VapW is a homolog of VapD without conserved catalytic residues required for nuclease activity. The crystal structure of the VapD-VapW complex reveals that VapW lacks the dimerization interface essential for the catalytic activity of VapD but retains the second interaction interface that enables VapD hexamerization. This allows VapW to bind VapD in the same manner that VapD dimers bind to each other in hexamers. Thus, though the VapD catalytic cleft remains accessible in the VapD-VapW complex, VapW may disrupt VapD oligomerization. To our knowledge, VapWD provides a unique example of TA systems evolution when a toxin loses its activity and becomes an antitoxin to itself.

Importance: Genes encoding virulence-associated protein D (VapD) homologs are found in many pathogens such as Helicobacter pylori, Haemophilus influenzae, and Xylella fastidiosa. There are many indications that VapD proteins contribute to virulence, even though the exact mechanism is not known. VapD proteins are either encoded by stand-alone genes or form toxin-antitoxin pairs with VapX. We performed a comprehensive census of vapD-like genes and found two new antitoxins, VapW and VapY. The VapW antitoxins are catalytically inactivated variants of VapD, revealing a new evolutionary mechanism for the appearance of toxin-antitoxin pairs.

Bacillus cereus, a Gram-positive aerobic bacterium commonly found in soil, food, and water, forms endospores that can withstand harsh environmental conditions. The endospores are encased in a protective spore coat consisting of multiple layers of proteins, among which, CotE serves as a crucial morphogenetic protein within the outer coat. In this study, we observed that the homotrimeric CotE protein underwent further oligomerization induced by Ca²⁺ and was subsequently dissociated by dipicolinic acid, a compound released from the spore core during germination. Through cryo-electron microscopy and tomography analyses of the Ca²⁺-induced CotE oligomer, combined with structural predictions and biochemical studies, we propose a three-dimensional meshwork organization facilitated by tryptophan-based interactions between CotE trimers. The resulting meshwork was organized in a defective diamond-like tetrahedral configuration. These insights enhance our understanding of how CotE contributes to endospore morphogenesis and germination through the rapid disassembly of these layers.

Importance: Bacterial endospores are highly resilient structures that allow bacteria to survive extreme environmental conditions, making them a significant concern in food safety and healthcare. The protein CotE plays a critical role in forming the protective outer coat of these endospores. Our research uncovers the three-dimensional meshwork architecture of CotE and reveals how it contributes to the structural integrity and rapid disassembly of endospores during germination. By understanding CotE's unique 3D structure and its interaction with other molecules, we gain valuable insights into how bacterial endospores are formed and how they can be effectively targeted for sterilization. This work not only advances our fundamental knowledge of bacterial endospore biology but also has potential applications in developing new strategies to combat bacterial contamination and improve sterilization techniques in the food and healthcare industries.