Capsid-dependent lentiviral restrictions.

Host antiviral proteins inhibit primate lentiviruses and other retroviruses by targeting many features of the viral life cycle. The lentiviral capsid protein and the assembled viral core are known to be inhibited through multiple, directly acting antiviral proteins. Several phenotypes, including those known as Lv1 through Lv5, have been described as cell type-specific blocks to infection against some but not all primate lentiviruses. Here we review important features of known capsid-targeting blocks to infection together with several blocks to infection for which the genes responsible for the inhibition still remain to be identified. We outline the features of these blocks as well as how current methodologies are now well suited to find these antiviral genes and solve these long-standing mysteries in the HIV and retrovirology fields.

Primate TRIM34 is a broadly-acting, TRIM5-dependent lentiviral restriction factor.

Human immunodeficiency virus (HIV) and other lentiviruses adapt to new hosts by evolving to evade host-specific innate immune proteins that differ in sequence and often viral recognition between host species. Understanding how these host antiviral proteins, called restriction factors, constrain lentivirus replication and transmission is key to understanding the emergence of pandemic viruses like HIV-1. Human TRIM34, a paralogue of the well-characterized lentiviral restriction factor TRIM5α, was previously identified by our lab via CRISPR-Cas9 screening as a restriction factor of certain HIV and SIV capsids. Here, we show that diverse primate TRIM34 orthologues from non-human primates can restrict a range of Simian Immunodeficiency Virus (SIV) capsids including SIVAGM-SAB, SIVAGM-TAN and SIVMAC capsids, which infect sabaeus monkeys, tantalus monkeys, and rhesus macaques, respectively. All primate TRIM34 orthologues tested, regardless of species of origin, were able to restrict this same subset of viral capsids. However, in all cases, this restriction also required the presence of TRIM5α. We demonstrate that TRIM5α is necessary, but not sufficient, for restriction of these capsids, and that human TRIM5α functionally interacts with TRIM34 from different species. Finally, we find that both the TRIM5α SPRY v1 loop and the TRIM34 SPRY domain are essential for TRIM34-mediated restriction. These data support a model in which TRIM34 is a broadly-conserved primate lentiviral restriction factor that acts in tandem with TRIM5α, such that together, these proteins can restrict capsids that neither can restrict alone.

Primate TRIM34 is a broadly-acting, TRIM5-dependent lentiviral restriction factor.

Human immunodeficiency virus (HIV) and other lentiviruses adapt to new hosts by evolving to evade host-specific innate immune proteins that differ in sequence and often viral recognition between host species. Understanding how these host antiviral proteins, called restriction factors, constrain lentivirus replication and transmission is key to understanding the emergence of pandemic viruses like HIV-1. Human TRIM34, a paralogue of the well-characterized lentiviral restriction factor TRIM5α, was previously identified by our lab via CRISPR-Cas9 screening as a restriction factor of certain HIV and SIV capsids. Here, we show that diverse primate TRIM34 orthologues from non-human primates can restrict a range of Simian Immunodeficiency Virus (SIV) capsids including SIV AGM-SAB , SIV AGM-TAN and SIV MAC capsids, which infect sabaeus monkeys, tantalus monkeys, and rhesus macaques, respectively. All primate TRIM34 orthologues tested, regardless of species of origin, were able to restrict this same subset of viral capsids. However, in all cases, this restriction also required the presence of TRIM5α. We demonstrate that TRIM5α is necessary, but not sufficient, for restriction of these capsids, and that human TRIM5α functionally interacts with TRIM34 from different species. Finally, we find that both the TRIM5α SPRY v1 loop and the TRIM34 SPRY domain are essential for TRIM34-mediated restriction. These data support a model in which TRIM34 is a broadly-conserved primate lentiviral restriction factor that acts in tandem with TRIM5α, such that together, these proteins can restrict capsids that neither can restrict alone.

Classical and γδ T cells are each independently sufficient to establish protection against a classical strain of Klebsiella pneumoniae.

Infections with classical strains of the Gram-negative bacterium Klebsiella pneumoniae pose a significant clinical challenge due to rising antibiotic resistance. We previously established a lung inoculation plus challenge model using live, classical K. pneumoniae in order to study host protection. Here, we employ this model to dissect adaptive immune responses to this critical pathogen. First, we performed convalescent serum transfers from inoculated mice to naïve recipients and found that classical K. pneumoniae infection outcomes, unlike hypervirulent K. pneumoniae infection outcomes, were not improved. This suggests that circulating antibody responses alone are not sufficient to mediate protection against this classical strain. Hence, we evaluated the role of T cells in protection against classical K. pneumoniae reinfection and demonstrated that mice lacking T cells are unable to establish a protective response. However, mice individually deficient in either of the major T cell subsets, γδ or αß (classical T cells), effectively mount a protective response, indicating either subset alone is sufficient to mediate protection. Sequestration of T cells in secondary lymphoid organs during the challenge infection did not ablate protection, indicating the circulating T cell pool is not required for the protective phenotype. Finally, we demonstrate that depletion of T cells during initial infection eliminates protection against challenge. Collectively, these experiments demonstrate the imperative contribution of T cells to protective immunity against classical K. pneumoniae and will guide further inquiries into host effector responses required to control this infection.

A murine model demonstrates capsule-independent adaptive immune protection in survivors of Klebsiella pneumoniae respiratory tract infection.

Klebsiella pneumoniae represents a growing clinical threat, given its rapid development of antibiotic resistance, necessitating new therapeutic strategies. Existing live-infection models feature high mortality rates, limiting their utility in the study of natural adaptive immune response to this pathogen. We developed a preclinical model of pneumonia with low overall mortality, in which previously exposed mice are protected from subsequent respiratory tract challenge with K. pneumoniae Histologic analyses of infected murine lungs demonstrate lymphocytic aggregates surrounding vasculature and larger airways. Initial exposure in RAG1 knockout mice (lacking functional B and T cells) failed to confer protection against subsequent K. pneumoniae challenge. While administration of isolated K. pneumoniae capsule was sufficient to provide protection, we also found that initial inoculation with K. pneumoniae mutants lacking capsule (Δcps), O-antigen (ΔwecA) or both conferred protection from subsequent wild-type infection and elicited K. pneumoniae-specific antibody responses, indicating that non-capsular antigens may also elicit protective immunity. Experiments in this model will inform future development of multivalent vaccines to prevent invasive K. pneumoniae infections.

TGFß1 orchestrates renal fibrosis following Escherichia coli pyelonephritis.

Renal scarring after pyelonephritis is linked to long-term health risks for hypertension and chronic kidney disease. Androgen exposure increases susceptibility to, and severity of, uropathogenic Escherichia coli (UPEC) pyelonephritis and resultant scarring in both male and female mice, while anti-androgen therapy is protective against severe urinary tract infection (UTI) in these models. This work employed androgenized female C57BL/6 mice to elucidate the molecular mechanisms of post-infectious renal fibrosis and to determine how these pathways are altered by the presence of androgens. We found that elevated circulating testosterone levels primed the kidney for fibrosis by increasing local production of TGFß1 before the initiation of UTI, altering the ratio of transcription factors Smad2 and Smad3 and increasing the presence of mesenchymal stem cell (MSC)-like cells and Gli1 + activated myofibroblasts, the cells primarily responsible for deposition of scar components. Increased production of TGFß1 and aberrations in Smad2:Smad3 were maintained throughout the course of infection in the presence of androgen, correlating with renal scarring that was not observed in non-androgenized female mice. Pharmacologic inhibition of TGFß1 signaling blunted myofibroblast activation. We conclude that renal fibrosis after pyelonephritis is exacerbated by the presence of androgens and involves activation of the TGFß1 signaling cascade, leading to increases in cortical populations of MSC-like cells and the Gli1 + activated myofibroblasts that are responsible for scarring.

A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) is globally disseminating as a community-acquired pathogen causing life-threatening infections in healthy individuals. The fact that a dose as little as 50 bacteria is lethal to mice illustrates the dramatic increase of virulence associated with hvKp strains compared with classical K. pneumoniae (cKp) strains, which require lethal doses greater than 107 bacteria. Until recently, these virulent strains were mostly antibiotic-susceptible. However, multidrug-resistant (MDR) hvKp strains have been emerging, spawning a new generation of hypervirulent "superbugs." The mechanisms of hypervirulence are not fully defined, but overproduction of capsular polysaccharide significantly impedes host clearance, resulting in increased pathogenicity of hvKp strains. While there are more than 80 serotypes of K. pneumoniae, the K1 and K2 serotypes cause the vast majority of hypervirulent infections. Therefore, a glycoconjugate vaccine targeting these 2 serotypes could significantly reduce hvKp infection. Conventionally, glycoconjugate vaccines are manufactured using intricate chemical methodologies to covalently attach purified polysaccharides to carrier proteins, which is widely considered to be technically challenging. Here we report on the recombinant production and analytical characterization of bioconjugate vaccines, enzymatically produced in glycoengineered Escherichia coli cells, against the 2 predominant hypervirulent K. pneumoniae serotypes, K1 and K2. The K. pneumoniae bioconjugates are immunogenic and efficacious, protecting mice against lethal infection from 2 hvKp strains, NTUH K-2044 and ATCC 43816. This preclinical study constitutes a key step toward preventing further global dissemination of hypervirulent MDR hvKp strains.

A non-canonical autophagy-dependent role of the ATG16L1T300A variant in urothelial vesicular trafficking and uropathogenic Escherichia coli persistence.

50% of Caucasians carry a Thr300Ala variant (T300A) in the protein encoded by the macroautophagy/autophagy gene ATG16L1. Here, we show that the T300A variant confers protection against urinary tract infections (UTIs), the most common infectious disease in women. Using knockin mice carrying the human T300A variant, we show that the variant limits the UTI-causing bacteria, uropathogenic Escherichia coli (UPEC), from establishing persistent intracellular reservoirs, which can seed UTI recurrence. This phenotype is recapitulated in mice lacking Atg16l1 or Atg7 exclusively in the urothelium. We further show that mice with the T300A variant exhibit urothelial cellular abnormalities, including vesicular congestion and aberrant accumulation of UPK (uroplakin) proteins. Importantly, presence of the T300A variant in humans is associated with similar urothelial architectural abnormalities, indicating an evolutionarily conserved impact. Mechanistically, we show that the reduced bacterial persistence is independent of basal autophagic flux or proinflammatory cytokine responses and does not involve Atg14 or Epg5. However, the T300A variant is associated with increased expression of the small GTPase Rab33b; RAB33B interacts with ATG16L1, as well as other secretory RABs, RAB27B and RAB11A, important for UPEC exocytosis from the urothelium. Finally, inhibition of secretory RABs in bladder epithelial cells increases intracellular UPEC load. Together, our results reveal that UPEC selectively utilize genes important for autophagosome formation to persist in the urothelium, and that the presence of the T300A variant in ATG16L1 is associated with changes in urothelial vesicle trafficking, which disrupts the ability of UPEC to persist, thereby limiting the risk of recurrent UTIs. Abbreviations: 3-PEHPC: 3-pyridinyl ethylidene hydroxyl phosphonocarboxylate; ATG: autophagy; ATG16L1: autophagy related 16 like 1; BECs: bladder epithelial cells; dpi: days post infection; hpi: hours post infection; IF: immunofluorescence; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MVB: multivesicular bodies; T300A: Thr300Ala; TNF: tumor necrosis factor; QIR(s): quiescent intracellular reservoir(s); siRNA: short interfering RNA; UPEC: uropathogenic Escherichia coli; UTI(s): urinary tract infection(s); TEM: transmission electron microscopy; WT: wild type.

Population Structure, Antibiotic Resistance, and Uropathogenicity of Klebsiella variicola.

Klebsiella variicola is a member of the Klebsiella genus and often misidentified as Klebsiella pneumoniae or Klebsiella quasipneumoniae The importance of K. pneumoniae human infections has been known; however, a dearth of relative knowledge exists for K. variicola Despite its growing clinical importance, comprehensive analyses of K. variicola population structure and mechanistic investigations of virulence factors and antibiotic resistance genes have not yet been performed. To address this, we utilized in silico, in vitro, and in vivo methods to study a cohort of K. variicola isolates and genomes. We found that the K. variicola population structure has two distant lineages composed of two and 143 genomes, respectively. Ten of 145 K. variicola genomes harbored carbapenem resistance genes, and 6/145 contained complete virulence operons. While the ß-lactam blaLEN and quinolone oqxAB antibiotic resistance genes were generally conserved within our institutional cohort, unexpectedly 11 isolates were nonresistant to the ß-lactam ampicillin and only one isolate was nonsusceptible to the quinolone ciprofloxacin. K. variicola isolates have variation in ability to cause urinary tract infections in a newly developed murine model, but importantly a strain had statistically significant higher bladder CFU than the model uropathogenic K. pneumoniae strain TOP52. Type 1 pilus and genomic identification of altered fim operon structure were associated with differences in bladder CFU for the tested strains. Nine newly reported types of pilus genes were discovered in the K. variicola pan-genome, including the first identified P-pilus in Klebsiella spp.IMPORTANCE Infections caused by antibiotic-resistant bacterial pathogens are a growing public health threat. Understanding of pathogen relatedness and biology is imperative for tracking outbreaks and developing therapeutics. Here, we detail the phylogenetic structure of 145 K. variicola genomes from different continents. Our results have important clinical ramifications as high-risk antibiotic resistance genes are present in K. variicola genomes from a variety of geographic locations and as we demonstrate that K. variicola clinical isolates can establish higher bladder titers than K. pneumoniae Differential presence of these pilus genes inK. variicola isolates may indicate adaption for specific environmental niches. Therefore, due to the potential of multidrug resistance and pathogenic efficacy, identification of K. variicola and K. pneumoniae to a species level should be performed to optimally improve patient outcomes during infection. This work provides a foundation for our improved understanding of K. variicola biology and pathogenesis.

In silico discovery of small molecules that inhibit RfaH recruitment to RNA polymerase.

RfaH is required for virulence in several Gram-negative pathogens including Escherichia coli and Klebsiella pneumoniae. Through direct interactions with RNA polymerase (RNAP) and ribosome, RfaH activates the expression of capsule, cell wall and pilus biosynthesis operons by reducing transcription termination and activating translation. While E. coli RfaH has been extensively studied using structural and biochemical approaches, limited data are available for other RfaH homologs. Here we set out to identify small molecule inhibitors of E. coli and K. pneumoniae RfaHs. Results of biochemical and functional assays show that these proteins act similarly, with a notable difference between their interactions with the RNAP ß subunit gate loop. We focused on high-affinity RfaH interactions with the RNAP ß' subunit clamp helices as a shared target for inhibition. Among the top 10 leads identified by in silico docking using ZINC database, 3 ligands were able to inhibit E. coli RfaH recruitment in vitro. The most potent lead was active against both E. coli and K. pneumoniae RfaHs in vitro. Our results demonstrate the feasibility of identifying RfaH inhibitors using in silico docking and pave the way for rational design of antivirulence therapeutics against antibiotic-resistant pathogens.

High Levels of Cyclic Di-GMP in Klebsiella pneumoniae Attenuate Virulence in the Lung.

The bacterial second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) has been shown to influence the expression of virulence factors in certain pathogenic bacteria, but little is known about its activity in the increasingly antibiotic-resistant pathogen Klebsiella pneumoniae Here, the expression in K. pneumoniae of a heterologous diguanylate cyclase increased the bacterial c-di-GMP concentration and attenuated pathogenesis in murine pneumonia. This attenuation remained evident in mice lacking the c-di-GMP sensor STING, indicating that the high c-di-GMP concentration exerted its influence not on host responses but on bacterial physiology. While serum resistance and capsule expression were unaffected by the increased c-di-GMP concentration, both type 3 and type 1 pili were strongly upregulated. Importantly, attenuation of K. pneumoniae virulence by high c-di-GMP levels was abrogated when type 1 pilus expression was silenced. We conclude that increased type 1 piliation may hamper K. pneumoniae virulence in the respiratory tract and that c-di-GMP signaling represents a potential therapeutic target for antibiotic-resistant K. pneumoniae in this niche.