The NSP3 protein of SARS-CoV-2 binds fragile X mental retardation proteins to disrupt UBAP2L interactions.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.

QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis.

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection.

Increased Seroprevalence of Typhus Group Rickettsiosis, Galveston County, Texas, USA.

Whether increases in typhus group rickettsiosis in Galveston County, Texas, USA, are caused by increased recognition or true reemergence is unclear. We conducted a serosurvey that demonstrated Rickettsia typhi antibodies increased from 1.2% in 2013 to 7.8% in 2021 (p<0.001). These findings support pathogen reemergence rather than enhanced recognition alone.

Serologic Evidence of Orientia Infection among Rural Population, Cauca Department, Colombia.

We assessed serum samples collected in Cauca Department, Colombia, from 486 persons for Orientia seroreactivity. Overall, 13.8% showed reactive IgG by indirect immunofluorescence antibody assay and ELISA. Of those samples, 30% (20/67) were confirmed to be positive by Western blot, showing >1 reactive band to Orientia 56-kD or 47-kD antigens.

Bartonella spp. and Typhus Group Rickettsiae among Persons Experiencing Homelessness, São Paulo, Brazil.

Persons experiencing homelessness in São Paulo, Brazil, were seropositive for Bartonella spp. (79/109, 72.5%) and typhus group rickettsiae (40/109, 36.7%). Bartonella quintana DNA was detected in 17.1% (14/82) body louse pools and 0.9% (1/114) blood samples. Clinicians should consider vectorborne agents as potential causes of febrile syndromes in this population.

Rickettsial Infections Causing Acute Febrile Illness in Urban Slums, Brazil.

We conducted enhanced acute febrile illness surveillance in an urban slum community in Salvador, Brazil. We found that rickettsial infection accounted for 3.5% of urgent care visits for acute fever. Our results suggest that rickettsiae might be an underrecognized, treatable cause of acute febrile illness in impoverished urban populations in Brazil.

Emerging Tickborne Bacteria in Cattle from Colombia.

Ehrlichia minasensis is a new pathogenic bacterial species that infects cattle, and Borrelia theileri causes bovine borreliosis. We detected E. minasensis and B. theileri DNA in cattle from southwestern Colombia by using PCR. E. minasensis and B. theileri should be considered potential etiologies of febrile syndrome in cattle from Colombia.

Immunoreactive Protein Repertoires of Ehrlichia chaffeensis and E. canis Reveal the Dominance of Hypothetical Proteins and Conformation-Dependent Antibody Epitopes.

The immunomes of Ehrlichia chaffeensis and Ehrlichia canis have recently been revised to include immunodominant hypothetical proteins with conformational antibody epitopes. In this study, we examined 216 E. chaffeensis and 190 E. canis highly antigenic proteins according to ANTIGENpro and also performed a genome-wide hypothetical protein analysis (E. chaffeensis n = 104; E. canis n = 124) for immunoreactivity. Using cell-free protein expression and immunoanalysis, 118 E. chaffeensis and 39 E. canis proteins reacted with sera from naturally E. chaffeensis-infected patients or E. canis-infected dogs. Moreover, 22 E. chaffeensis and 18 E. canis proteins consistently and strongly reacted with a panel of patient or canine sera. A subset of E. chaffeensis (n = 18) and E. canis (n = 9) proteins were identified as immunodominant. Consistent with our previous study, most proteins were classified as hypothetical, and the antibody epitopes exhibited complete or partial conformation dependence. The majority (28/40, 70%) of E. chaffeensis and E. canis proteins contained transmembrane domains, and 19 (48%) were predicted to be secreted effectors. The antigenic repertoires of E. chaffeensis and E. canis were mostly diverse and suggest that the immunomes of these closely related ehrlichiae are dominated by species-specific conformational antibody epitopes. This study reveals a significant group of previously undefined E. chaffeensis and E. canis antigens and reaffirms the importance of conformation-dependent epitopes as targets of anti-Ehrlichia immune responses. These findings substantially expand our understanding of host-Ehrlichia immune responses, advance efforts to define the molecular features of protective proteins, and improve prospects for effective vaccines for the ehrlichioses.

Quantitative Proteomics of the Endothelial Secretome Identifies RC0497 as Diagnostic of Acute Rickettsial Spotted Fever Infections.

Mediterranean spotted fever is a reemerging acute tick-borne infection produced by the α-proteobacterium, Rickettsia conorii. Rickettsia conorii infects vascular endothelial cells producing disseminated plasma leakage, manifesting as nonspecific fever, headache, and maculopapular rash. Because there are no available tests of early infection, Mediterranean spotted fever is often undiagnosed and untreated, resulting in significant mortality. To address this critical need, we have applied a quantitative proteomics pipeline for analyzing the secretome of primary human umbilical vein endothelial cells. Of the 104 proteins whose abundance changed significantly in the R. conorii-infected human umbilical vein endothelial cells' secretome, 46 proteins were up-regulated: 45 were host secreted proteins (including cytokines), and 1 was a rickettsial protein, the putative N-acetylmuramoyl-l-alanine amidase RC0497. Proteins with sequence highly homologous to RC0497 were found to be shared by many species of the spotted fever group rickettsiae, but not typhus group rickettsiae. Quantitative targeted proteomics studies of plasma from a mouse model of sublethal and lethal R. conorii identified RC0497 in the blood, and its circulating levels were proportionally associated with infection outcome. Finally, the presence of RC0497 in the serum samples from a cohort of humans presenting with acute rickettsioses was confirmed. The detection of RC0497 has the potential to be a sensitive and specific marker for acute rickettsial spotted rickettsioses.

Activation of ASC Inflammasome Driven by Toll-Like Receptor 4 Contributes to Host Immunity against Rickettsial Infection.

Rickettsiae are cytosolically replicating, obligately intracellular bacteria causing human infections worldwide with potentially fatal outcomes. We previously showed that Rickettsia australis activates ASC inflammasome in macrophages. In the present study, host susceptibility of ASC inflammasome-deficient mice to R. australis was significantly greater than that of C57BL/6 (B6) controls and was accompanied by increased rickettsial loads in various organs. Impaired host control of R. australis in vivo in ASC-/- mice was associated with dramatically reduced levels of interleukin 1ß (IL-1ß), IL-18, and gamma interferon (IFN-γ) in sera. The intracellular concentrations of R. australis in bone marrow-derived macrophages (BMMs) of TLR4-/- and ASC-/- mice were significantly greater than those in BMMs of B6 controls, highlighting the important role of inflammasome and these molecules in controlling rickettsiae in macrophages. Compared to B6 BMMs, TLR4-/- BMMs failed to secrete a significant level of IL-1ß and had reduced expression levels of pro-IL-1ß in response to infection with R. australis, suggesting that rickettsiae activate ASC inflammasome via a Toll-like receptor 4 (TLR4)-dependent mechanism. Further mechanistic studies suggest that the lipopolysaccharide (LPS) purified from R. australis together with ATP stimulation led to cleavage of pro-caspase-1 and pro-IL-1ß, resulting in TLR4-dependent secretion of IL-1ß. Taken together, these observations indicate that activation of ASC inflammasome, most likely driven by interaction of TLR4 with rickettsial LPS, contributes to host protective immunity against R. australis These findings provide key insights into defining the interactions of rickettsiae with the host innate immune system.

Burkholderia pseudomallei as an Enteric Pathogen: Identification of Virulence Factors Mediating Gastrointestinal Infection.

Burkholderia pseudomallei is a Gram-negative bacterium and the causative agent of melioidosis. Despite advances in our understanding of the disease, B. pseudomallei poses a significant health risk, especially in regions of endemicity, where treatment requires prolonged antibiotic therapy. Even though the respiratory and percutaneous routes are well documented and considered the main ways to acquire the pathogen, the gastrointestinal tract is believed to be an underreported and underrecognized route of infection. In the present study, we describe the development of in vitro and in vivo models to study B. pseudomallei gastrointestinal infection. Further, we report that the type 6 secretion system (T6SS) and type 1 fimbriae are important virulence factors required for gastrointestinal infection. Using a human intestinal epithelial cell line and mouse primary intestinal epithelial cells (IECs), we demonstrated that B. pseudomallei adheres, invades, and forms multinucleated giant cells, ultimately leading to cell toxicity. We demonstrated that mannose-sensitive type 1 fimbria is involved in the initial adherence of B. pseudomallei to IECs, although the impact on full virulence was limited. Finally, we also showed that B. pseudomallei requires a functional T6SS for full virulence, bacterial dissemination, and lethality in mice infected by the intragastric route. Overall, we showed that B. pseudomallei is an enteric pathogen and that type 1 fimbria is important for B. pseudomallei intestinal adherence, and we identify a new role for T6SS as a key virulence factor in gastrointestinal infection. These studies highlight the importance of gastrointestinal melioidosis as an understudied route of infection and open a new avenue for the pathogenesis of B. pseudomallei.

Human Rickettsiosis Caused by Rickettsia parkeri Strain Atlantic Rainforest, Urabá, Colombia.

We describe the clinical, serologic, and molecular findings of a new human rickettsiosis in Colombia. Antibodies against Rickettsia spp. were detected. PCR showed amplification of genes for R. parkeri strain Atlantic Rainforest. This new rickettsiosis of minor virulence could explain some of the undifferentiated acute febrile diseases in Colombia.

Human Monocytic Ehrlichiosis, Mexico City, Mexico.

Little information is available about human infections by the members of the genus Ehrlichia in Mexico. Only 2 species, Ehrlichia canis and E. chaffensis, are known to cause disease in this country. We report a fatal case of human monocytic ehrlichiosis in Mexico City in a man who was homeless.

Vector Tick Transmission Model of Spotted Fever Rickettsiosis.

Many aspects of rickettsial infections have been characterized, including pathogenic and immune pathways and mechanisms of rickettsial survival within the vertebrate host and tick vector. However, very few studies are focused on the complex pathogen-vector-host interactions during tick feeding. Therefore, our objective was to develop a tick transmission model of the spotted fever group of rickettsial infections to study the initial events in disease development. The most appropriate strain of mouse was identified for evaluation as a transmission model, and the course of infection, bacterial levels, histopathologic changes, and antibody response during tick transmission in mice infested with Amblyomma maculatum ticks carrying the emerging pathogen, Rickettia parkeri, were studied. Results showed distinct clinical signs in C3H/HeN mice infected intravenously, leading to selection of this mouse strain for tick transmission studies. Active infection of animals was observed after tick vector transmission. The bacteria disseminated systemically and spread to several organs at 24 hours after tick attachment, with peak bacterial load at day 6 after tick attachment. Skin, lung, and liver showed the greatest pathologic changes, with inflammatory cellular infiltration and necrosis. These findings indicate the feasibility of using murine infection with R. parkeri by A. maculatum tick transmission as a model to study different aspects of the spotted fever group of rickettsial disease establishment.

Ehrlichia chaffeensis Outer Membrane Protein 1-Specific Human Antibody-Mediated Immunity Is Defined by Intracellular TRIM21-Dependent Innate Immune Activation and Extracellular Neutralization.

Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.

Atg5 Supports Rickettsia australis Infection in Macrophages In Vitro and In Vivo.

Rickettsiae can cause life-threatening infections in humans. Macrophages are one of the initial targets for rickettsiae after inoculation by ticks. However, it remains poorly understood how rickettsiae remain free in macrophages prior to establishing their infection in microvascular endothelial cells. Here, we demonstrated that the concentration of Rickettsia australis was significantly greater in infected tissues of Atg5flox/flox mice than in the counterparts of Atg5flox/flox Lyz-Cre mice, in association with a reduced level of interleukin-1ß (IL-1ß) in serum. The greater concentration of R. australis in Atg5flox/flox bone marrow-derived macrophages (BMMs) than in Atg5flox/flox Lyz-Cre BMMs in vitro was abolished by exogenous treatment with recombinant IL-1ß. Rickettsia australis induced significantly increased levels of light chain 3 (LC3) form II (LC3-II) and LC3 puncta in Atg5-competent BMMs but not in Atg5-deficient BMMs, while no p62 turnover was observed. Further analysis found the colocalization of LC3 with a small portion of R. australis and Rickettsia-containing double-membrane-bound vacuoles in the BMMs of B6 mice. Moreover, treatment with rapamycin significantly increased the concentrations of R. australis in B6 BMMs compared to those in the untreated B6 BMM controls. Taken together, our results demonstrate that Atg5 favors R. australis infection in mouse macrophages in association with a suppressed level of IL-1ß production but not active autophagy flux. These data highlight the contribution of Atg5 in macrophages to the pathogenesis of rickettsial diseases.

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections.