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A comparative study of in vitro air-liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution

Prescott, Rachel A; Pankow, Alec P; de Vries, Maren; Crosse, Keaton M; Patel, Roosheel S; Alu, Mark; Loomis, Cynthia; Torres, Victor; Koralov, Sergei; Ivanova, Ellie; Dittmann, Meike; Rosenberg, Brad R
BACKGROUND:The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial (HAE) cultures at air-liquid interface are a physiologically relevant in vitro model of this heterogeneous tissue and have enabled numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining the capacity for differentiation to HAE. However, gene expression and innate immune function in BCi-NS1.1-derived versus primary-derived HAE cultures have not been fully characterized. METHODS:BCi-NS1.1-derived HAE cultures (n = 3 independent differentiations) and primary-derived HAE cultures (n = 3 distinct donors) were characterized by immunofluorescence and single cell RNA-Seq (scRNA-Seq). Innate immune functions were evaluated in response to interferon stimulation and to infection with viral and bacterial respiratory pathogens. RESULTS:We confirm at high resolution that BCi-NS1.1- and primary-derived HAE cultures are largely similar in morphology, cell type composition, and overall gene expression patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1-derived HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. CONCLUSIONS:Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.
PMID: 37635251
ISSN: 1465-993x
CID: 5606922

Rewilding of laboratory mice enhances granulopoiesis and immunity through intestinal fungal colonization

Chen, Ying-Han; Yeung, Frank; Lacey, Keenan A; Zaldana, Kimberly; Lin, Jian-Da; Bee, Gavyn Chern Wei; McCauley, Caroline; Barre, Ramya S; Liang, Shen-Huan; Hansen, Christina B; Downie, Alexander E; Tio, Kyle; Weiser, Jeffrey N; Torres, Victor J; Bennett, Richard J; Loke, P'ng; Graham, Andrea L; Cadwell, Ken
The paucity of blood granulocyte populations such as neutrophils in laboratory mice is a notable difference between this model organism and humans, but the cause of this species-specific difference is unclear. We previously demonstrated that laboratory mice released into a seminatural environment, referred to as rewilding, display an increase in blood granulocytes that is associated with expansion of fungi in the gut microbiota. Here, we find that tonic signals from fungal colonization induce sustained granulopoiesis through a mechanism distinct from emergency granulopoiesis, leading to a prolonged expansion of circulating neutrophils that promotes immunity. Fungal colonization after either rewilding or oral inoculation of laboratory mice with Candida albicans induced persistent expansion of myeloid progenitors in the bone marrow. This increase in granulopoiesis conferred greater long-term protection from bloodstream infection by gram-positive bacteria than by the trained immune response evoked by transient exposure to the fungal cell wall component β-glucan. Consequently, introducing fungi into laboratory mice may restore aspects of leukocyte development and provide a better model for humans and free-living mammals that are constantly exposed to environmental fungi.
PMID: 37352372
ISSN: 2470-9468
CID: 5537252

Secreted mammalian DNases protect against systemic bacterial infection by digesting biofilms

Lacey, Keenan A; Serpas, Lee; Makita, Sohei; Wang, Yueyang; Rashidfarrokhi, Ali; Soni, Chetna; Gonzalez, Sandra; Moreira, Andre; Torres, Victor J; Reizis, Boris
Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.
PMID: 36928522
ISSN: 1540-9538
CID: 5449012

Multivalent human antibody-centyrin fusion protein to prevent and treat Staphylococcus aureus infections

Buckley, Peter T; Chan, Rita; Fernandez, Jeffrey; Luo, Jinquan; Lacey, Keenan A; DuMont, Ashley L; O'Malley, Aidan; Brezski, Randall J; Zheng, Songmao; Malia, Thomas; Whitaker, Brian; Zwolak, Adam; Payne, Angela; Clark, Desmond; Sigg, Martin; Lacy, Eilyn R; Kornilova, Anna; Kwok, Debra; McCarthy, Steve; Wu, Bingyuan; Morrow, Brian; Nemeth-Seay, Jennifer; Petley, Ted; Wu, Sam; Strohl, William R; Lynch, Anthony Simon; Torres, Victor J
Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein ("mAbtyrin") that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.
PMID: 37098341
ISSN: 1934-6069
CID: 5465112

SarS Is a Repressor of Staphylococcus aureus Bicomponent Pore-Forming Leukocidins

Anderson, Exene E; Dyzenhaus, Sophie; Ilmain, Juliana K; Sullivan, Mitchell J; van Bakel, Harm; Torres, Victor J
Staphylococcus aureus is a successful pathogen that produces a wide range of virulence factors that it uses to subvert and suppress the immune system. These include the bicomponent pore-forming leukocidins. How the expression of these toxins is regulated is not completely understood. Here, we describe a screen to identify transcription factors involved in the regulation of leukocidins. The most prominent discovery from this screen is that SarS, a known transcription factor which had previously been described as a repressor of alpha-toxin expression, was found to be a potent repressor of leukocidins LukED and LukSF-PV. We found that inactivating sarS resulted in increased virulence both in an ex vivo model using primary human neutrophils and in an in vivo infection model in mice. Further experimentation revealed that SarS represses leukocidins by serving as an activator of Rot, a critical repressor of toxins, as well as by directly binding and repressing the leukocidin promoters. By studying contemporary clinical isolates, we identified naturally occurring mutations in the sarS promoter that resulted in overexpression of sarS and increased repression of leukocidins in USA300 bloodstream clinical isolates. Overall, these data establish SarS as an important repressor of leukocidins and expand our understanding of how these virulence factors are being regulated in vitro and in vivo by S. aureus.
PMID: 36939325
ISSN: 1098-5522
CID: 5462722

Regulation of Bacterial Two-Component Systems by Cardiolipin

Yeo, Won-Sik; Dyzenhaus, Sophie; Torres, Victor J; Brinsmade, Shaun R; Bae, Taeok
The regulation of membrane protein activity for cellular functions is critically dependent on the composition of phospholipid membranes. Cardiolipin, a unique phospholipid found in bacterial membranes and mitochondrial membranes of eukaryotes, plays a crucial role in stabilizing membrane proteins and maintaining their function. In the human pathogen Staphylococcus aureus, the SaeRS two-component system (TCS) controls the expression of key virulence factors essential for the bacterium's virulence. The SaeS sensor kinase activates the SaeR response regulator via phosphoryl transfer to bind its gene target promoters. In this study, we report that cardiolipin is critical for sustaining the full activity of SaeRS and other TCSs in S. aureus. The sensor kinase protein SaeS binds directly to cardiolipin and phosphatidylglycerol, enabling SaeS activity. Elimination of cardiolipin from the membrane reduces SaeS kinase activity, indicating that bacterial cardiolipin is necessary for modulating the kinase activities of SaeS and other sensor kinases during infection. Moreover, the deletion of cardiolipin synthase genes cls1 and cls2 leads to reduced cytotoxicity to human neutrophils and lower virulence in a mouse model of infection. These findings suggest a model where cardiolipin modulates the kinase activity of SaeS and other sensor kinases after infection to adapt to the hostile environment of the host and expand our knowledge of how phospholipids contribute to membrane protein function.
PMID: 36975788
ISSN: 1098-5522
CID: 5463172

Capsular Polysaccharide Is Essential for the Virulence of the Antimicrobial-Resistant Pathogen Enterobacter hormaechei

St John, Amelia; Perault, Andrew I; Giacometti, Sabrina I; Sommerfield, Alexis G; DuMont, Ashley L; Lacey, Keenan A; Zheng, Xuhui; Sproch, Julia; Petzold, Chris; Dancel-Manning, Kristen; Gonzalez, Sandra; Annavajhala, Medini; Beckford, Colleen; Zeitouni, Nathalie; Liang, Feng-Xia; van Bakel, Harm; Shopsin, Bo; Uhlemann, Anne-Catrin; Pironti, Alejandro; Torres, Victor J
Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.
PMID: 36779722
ISSN: 2150-7511
CID: 5421192

MRSA lineage USA300 isolated from bloodstream infections exhibit altered virulence regulation

Dyzenhaus, Sophie; Sullivan, Mitchell J; Alburquerque, Bremy; Boff, Daiane; van de Guchte, Adriana; Chung, Marilyn; Fulmer, Yi; Copin, Richard; Ilmain, Juliana K; O'Keefe, Anna; Altman, Deena R; Stubbe, François-Xavier; Podkowik, Magdalena; Dupper, Amy C; Shopsin, Bo; van Bakel, Harm; Torres, Victor J
The epidemic community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 lineage has recently become a leading cause of hospital-associated bloodstream infections (BSIs). Here, we leveraged this recent introduction into hospitals and the limited genetic variation across USA300 isolates to identify mutations that contribute to its success in a new environment. We found that USA300 BSI isolates exhibit altered virulence regulation. Using comparative genomics to delineate the genes involved in this phenotype, we discovered repeated and independent mutations in the transcriptional regulator sarZ. Mutations in sarZ resulted in increased virulence of USA300 BSI isolates in a murine model of BSI. The sarZ mutations derepressed the expression and production of the surface protein ClfB, which was critical for the pathogenesis of USA300 BSI isolates. Altogether, these findings highlight ongoing evolution of a major MRSA lineage and suggest USA300 strains can optimize their fitness through altered regulation of virulence.
PMID: 36681080
ISSN: 1934-6069
CID: 5426472

Endothelial ACKR1 is induced by neutrophil contact and down-regulated by secretion in extracellular vesicles

Guo, Xinying; Khosraviani, Negar; Raju, Sneha; Singh, Joshya; Farahani, Nikki Zamani; Abramian, Madlene; Torres, Victor J; Howe, Kathryn L; Fish, Jason E; Kapus, Andras; Lee, Warren L
Atypical chemokine receptor-1 (ACKR1), previously known as the Duffy antigen receptor for chemokines, is a widely conserved cell surface protein that is expressed on erythrocytes and the endothelium of post-capillary venules. In addition to being the receptor for the parasite causing malaria, ACKR1 has been postulated to regulate innate immunity by displaying and trafficking chemokines. Intriguingly, a common mutation in its promoter leads to loss of the erythrocyte protein but leaves endothelial expression unaffected. Study of endothelial ACKR1 has been limited by the rapid down-regulation of both transcript and protein when endothelial cells are extracted and cultured from tissue. Thus, to date the study of endothelial ACKR1 has been limited to heterologous over-expression models or the use of transgenic mice. Here we report that exposure to whole blood induces ACKR1 mRNA and protein expression in cultured primary human lung microvascular endothelial cells. We found that contact with neutrophils is required for this effect. We show that NF-κB regulates ACKR1 expression and that upon removal of blood, the protein is rapidly secreted by extracellular vesicles. Finally, we confirm that endogenous ACKR1 does not signal upon stimulation with IL-8 or CXCL1. Our observations define a simple method for inducing endogenous endothelial ACKR1 protein that will facilitate further functional studies.
PMID: 37153544
ISSN: 1664-3224
CID: 5503252

Clostridia isolated from helminth-colonized humans promote the life cycle of Trichuris species

Sargsian, Shushan; Chen, Ze; Lee, Soo Ching; Robertson, Amicha; Thur, Rafaela Saes; Sproch, Julia; Devlin, Joseph C; Tee, Mian Zi; Er, Yi Xian; Copin, Richard; Heguy, Adriana; Pironti, Alejandro; Torres, Victor J; Ruggles, Kelly V; Lim, Yvonne A L; Bethony, Jeffrey; Loke, P'ng; Cadwell, Ken
Soil-transmitted intestinal worms known as helminths colonize over 1.5 billion people worldwide. Although helminth colonization has been associated with altered composition of the gut microbiota, such as increases in Clostridia, individual species have not been isolated and characterized. Here, we isolate and sequence the genome of 13 Clostridia from the Orang Asli, an indigenous population in Malaysia with a high prevalence of helminth infections. Metagenomic analysis of 650 fecal samples from urban and rural Malaysians confirm the prevalence of species corresponding to these isolates and reveal a specific association between Peptostreptococcaceae family members and helminth colonization. Remarkably, Peptostreptococcaceae isolated from the Orang Asli display superior capacity to promote the life cycle of whipworm species, including hatching of eggs from Trichuris muris and Trichuris trichiura. These findings support a model in which helminths select for gut colonization of microbes that support their life cycle.
PMID: 36450245
ISSN: 2211-1247
CID: 5374022