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COVID-19 presents with a plethora of neurological signs and symptoms despite being characterized as a respiratory disease, including seizures, anxiety, depression, amnesia, attention deficits, and alterations in consciousness. The olfactory nerve is widely accepted as the neuroinvasive route by which the etiological agent SARS-CoV-2 enters the brain, but the trigeminal nerve is an often-overlooked additional route. Based on this consensus, we initially conducted a pilot experiment investigating the olfactory nerve route of SARS-CoV-2 neuroinvasion via intranasal inoculation in AC70 human ACE2 transgenic mice. Notably, we found that the trigeminal ganglion is an early and highly efficient site of viral replication, which then rapidly spread widely throughout the brain where neurons were primarily targeted. Despite the extensive viral infection across the brain, obvious evidence of tissue pathology including inflammatory infiltration, glial activation, and apoptotic cell deaths were not consistently observed, albeit inflammatory cytokines were significantly induced. However, the expression levels of different genes related to neuronal function, including the neurotransmitter dopamine pathway as well as synaptic function, and markers of neuronal damage were altered as compared to mock-infected mice. Our findings suggest that the trigeminal nerve may serve as a neuroinvasive route complementary to the olfactory nerve and that the ensuing neuroinvasion presented a unique neuropathological profile. This study provides insights into potential neuropathogenic mechanisms utilized by coronaviruses.

COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as ~ 3 and ~ 0.5 TCID50 of SARS-CoV-2, respectively. Mice infected with 105 TCID50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and inflammatory mediators could be detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio (NLR). Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy. SARS-CoV-2 could induce the release of circulating neutrophil extracellular traps (NETs), along with activated platelet/endothelium marker. Immunohistochemical staining with anti-platelet factor-4 (PF4) antibody revealed profound platelet aggregates especially within blocked veins of the lungs. We showed that acute SARS-CoV-2 infection triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis. Finally, we highlighted the potential role of Annexin A2 (ANXA2) in fibrinolytic failure. ANXA2 is a calcium-dependent phospholipid-binding protein that forms a heterotertrameric complexes localized at the extracellular membranes with two S100A10 small molecules acting as a co-receptor for tissue-plasminogen activator (t-PA), tightly involved in cell surface fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced ANXA2/S100A10 association in infected mice, this protein might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19.

ABSTRACTUrinary tract infection (UTI) is a ubiquitous infectious condition, and uropathogenic Escherichia coli (UPEC) is the predominant causative agent of UTI. Copper (Cu) is implicated in innate immunity, including against UPEC. Cu is a trace element utilized as a co-factor, but excess Cu is toxic due to mismetalation of non-cognate proteins. E. coli precisely regulates Cu homeostasis via efflux systems. However, Cu import mechanisms into the bacterial cell are not clear. We hypothesized that Cu import defective mutants would exhibit increased resistance to Cu. This hypothesis was tested in a forward genetic screen with transposon (Tn5) insertion mutants in UPEC strain CFT073, and we identified 32 unique Cu-resistant mutants. Transposon and defined mutants lacking yhiM, which encodes a hypothetical inner membrane protein, were more resistant to Cu than parental strain. Loss of YhiM led to decreased cellular Cu content and increased expression of copA, encoding a Cu efflux pump. The CpxAR envelope stress response system was activated in the ΔyhiM mutant as indicated by increased expression of cpxP. Transcription of yhiM was regulated by CueR and CpxR, and the CpxAR system was essential for increased Cu resistance in the ΔyhiM mutant. Importantly, activation of CpxAR system in the ΔyhiM mutant was independent of NlpE, a known activator of this system. YhiM was required for optimal fitness of UPEC in a mouse model of UTI. Our findings demonstrate that YhiM is a critical mediator of Cu homeostasis and links bacterial adaptation to Cu stress with the CpxAR-dependent envelope stress response in UPEC.IMPORTANCEUPEC is a common bacterial infection. Bacterial pathogens are exposed to host-derived Cu during infection, including UTI. Here, we describe detection of genes involved in Cu homeostasis in UPEC. A UPEC mutant lacking YhiM, a membrane protein, exhibited dramatic increase in resistance to Cu. Our study demonstrates YhiM as a nexus between Cu stress and the CpxAR-dependent envelope stress response system. Importantly, our findings establish NlpE-independent activation of CpxAR system during Cu stress in UPEC. Collectively, YhiM emerges as a critical mediator of Cu homeostasis in UPEC and highlights the interlinked nature of bacterial adaptation to survival during Cu and envelope stress.

Licensed COVID-19 vaccines ameliorate viral infection by inducing production of neutralizing antibodies that bind the SARS-CoV-2 Spike protein and inhibit viral cellular entry. However, the clinical effectiveness of these vaccines is transitory as viral variants escape antibody neutralization. Effective vaccines that solely rely upon a T cell response to combat SARS-CoV-2 infection could be transformational because they can utilize highly conserved short pan-variant peptide epitopes, but a mRNA-LNP T cell vaccine has not been shown to provide effective anti-SARS-CoV-2 prophylaxis. Here we show a mRNA-LNP vaccine (MIT-T-COVID) based on highly conserved short peptide epitopes activates CD8+ and CD4+ T cell responses that attenuate morbidity and prevent mortality in HLA-A*02:01 transgenic mice infected with SARS-CoV-2 Beta (B.1.351). We found CD8+ T cells in mice immunized with MIT-T-COVID vaccine significantly increased from 1.1% to 24.0% of total pulmonary nucleated cells prior to and at 7 days post infection (dpi), respectively, indicating dynamic recruitment of circulating specific T cells into the infected lungs. Mice immunized with MIT-T-COVID had 2.8 (2 dpi) and 3.3 (7 dpi) times more lung infiltrating CD8+ T cells than unimmunized mice. Mice immunized with MIT-T-COVID had 17.4 times more lung infiltrating CD4+ T cells than unimmunized mice (7 dpi). The undetectable specific antibody response in MIT-T-COVID-immunized mice demonstrates specific T cell responses alone can effectively attenuate the pathogenesis of SARS-CoV-2 infection. Our results suggest further study is merited for pan-variant T cell vaccines, including for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID.

ABSTRACT Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly-N-substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (

Urinary tract infection (UTI) is an extremely common infectious condition affecting people throughout the world. Increasing antibiotic resistance in pathogens causing UTI threatens our ability to continue to treat patients in the clinics.

Up to one-third of individuals suffering from acute SARS-CoV-2 infection with the onset of severe-to-mild diseases could develop several symptoms of neurological disorders, which could last long after resolving the infection, known as neuro-COVID. Effective therapeutic treatments for neuro-COVID remain unavailable, in part, due to the absence of animal models for studying its underlying mechanisms and developing medical countermeasures against it. Here, we explored the impact of SARS-CoV-2 infection on the well-being of respiratory and neurological functions of BALB/c mice by using a clinical isolate of β-variant, i.e. B.1.351. We found that this β-variant of SARS-CoV-2 primarily infected the lungs, causing tissue damage, profound inflammatory responses, altered respiratory functions and transient but significant hypoxia. Although live progeny viruses could not be isolated, viral RNAs were detected across many anatomical regions of the brains in most challenged mice and triggered activation of genes encoding for NF -kB , IL-6 , IP-10 and RANTES and microglial cells. We noted that the significantly activated IL-6 -encoded gene persisted at 4 weeks after infection. Together, these results suggest that this B.1.351/BALB/c model of SARS-CoV-2 infection warrants further studies to establish it as a desirable model for studies of neuropathogenesis and the development of effective therapeutics of neuro-COVID.

COVID-19 presents with a plethora of neurological signs and symptoms despite being characterized as a respiratory disease, including seizures, anxiety, depression, amnesia, attention deficits, and alterations in consciousness. The olfactory nerve is widely accepted as the neuroinvasive route by which the etiological agent SARS-CoV-2 enters the brain, but the trigeminal nerve is an often-overlooked additional route. Based on this consensus, we initially conducted a pilot experiment investigating the olfactory nerve route of SARS-CoV-2 neuroinvasion via intranasal inoculation in AC70 human ACE2 transgenic mice. Notably, we found that the trigeminal ganglion is an early and highly efficient site of viral replication, which then rapidly spread widely throughout the brain where neurons were primarily targeted. Despite the extensive viral infection across the brain, obvious evidence of tissue pathology including inflammatory infiltration, glial activation, and apoptotic cell deaths were not consistently observed, albeit inflammatory cytokines were significantly induced. However, the expression levels of different genes related to neuronal function, including the neurotransmitter dopamine pathway as well as synaptic function, and markers of neuronal damage were altered as compared to mock-infected mice. Our findings suggest that the trigeminal nerve may serve as a neuroinvasive route complementary to the olfactory nerve and that the ensuing neuroinvasion presented a unique neuropathological profile. This study provides insights into potential neuropathogenic mechanisms utilized by coronaviruses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hsu, Saenkham-Huntsinger, Huang, Octaviani, Drelich, Peng and Tseng.)

COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as ~ 3 and ~ 0.5 TCID50 of SARS-CoV-2, respectively. Mice infected with 105 TCID50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and inflammatory mediators could be detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio (NLR). Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy. SARS-CoV-2 could induce the release of circulating neutrophil extracellular traps (NETs), along with activated platelet/endothelium marker. Immunohistochemical staining with anti-platelet factor-4 (PF4) antibody revealed profound platelet aggregates especially within blocked veins of the lungs. We showed that acute SARS-CoV-2 infection triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis. Finally, we highlighted the potential role of Annexin A2 (ANXA2) in fibrinolytic failure. ANXA2 is a calcium-dependent phospholipid-binding protein that forms a heterotertrameric complexes localized at the extracellular membranes with two S100A10 small molecules acting as a co-receptor for tissue-plasminogen activator (t-PA), tightly involved in cell surface fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced ANXA2/S100A10 association in infected mice, this protein might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Drelich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Urinary tract infection (UTI) is a ubiquitous infectious condition, and uropathogenic Escherichia coli (UPEC) is the predominant causative agent of UTI. Copper (Cu) is implicated in innate immunity, including against UPEC. Cu is a trace element utilized as a co-factor, but excess Cu is toxic due to mismetalation of non-cognate proteins. E. coli precisely regulates Cu homeostasis via efflux systems. However, Cu import mechanisms into the bacterial cell are not clear. We hypothesized that Cu import defective mutants would exhibit increased resistance to Cu. This hypothesis was tested in a forward genetic screen with transposon (Tn 5 ) insertion mutants in UPEC strain CFT073, and we identified 32 unique Cu-resistant mutants. Transposon and defined mutants lacking yhiM , which encodes a hypothetical inner membrane protein, were more resistant to Cu than parental strain. Loss of YhiM led to decreased cellular Cu content and increased expression of copA , encoding a Cu efflux pump. The CpxAR envelope stress response system was activated in the ΔyhiM mutant as indicated by increased expression of cpxP . Transcription of yhiM was regulated by CueR and CpxR, and the CpxAR system was essential for increased Cu resistance in the ΔyhiM mutant. Importantly, activation of CpxAR system in the ΔyhiM mutant was independent of NlpE, a known activator of this system. YhiM was required for optimal fitness of UPEC in a mouse model of UTI. Our findings demonstrate that YhiM is a critical mediator of Cu homeostasis and links bacterial adaptation to Cu stress with the CpxAR-dependent envelope stress response in UPEC.IMPORTANCEUPEC is a common bacterial infection. Bacterial pathogens are exposed to host-derived Cu during infection, including UTI. Here, we describe detection of genes involved in Cu homeostasis in UPEC. A UPEC mutant lacking YhiM, a membrane protein, exhibited dramatic increase in resistance to Cu. Our study demonstrates YhiM as a nexus between Cu stress and the CpxAR-dependent envelope stress response system. Importantly, our findings establish NlpE-independent activation of CpxAR system during Cu stress in UPEC. Collectively, YhiM emerges as a critical mediator of Cu homeostasis in UPEC and highlights the interlinked nature of bacterial adaptation to survival during Cu and envelope stress., Competing Interests: The authors declare no conflict of interest.

Licensed COVID-19 vaccines ameliorate viral infection by inducing production of neutralizing antibodies that bind the SARS-CoV-2 Spike protein and inhibit viral cellular entry. However, the clinical effectiveness of these vaccines is transitory as viral variants escape antibody neutralization. Effective vaccines that solely rely upon a T cell response to combat SARS-CoV-2 infection could be transformational because they can utilize highly conserved short pan-variant peptide epitopes, but a mRNA-LNP T cell vaccine has not been shown to provide effective anti-SARS-CoV-2 prophylaxis. Here we show a mRNA-LNP vaccine (MIT-T-COVID) based on highly conserved short peptide epitopes activates CD8 + and CD4 + T cell responses that attenuate morbidity and prevent mortality in HLA-A*02:01 transgenic mice infected with SARS-CoV-2 Beta (B.1.351). We found CD8 + T cells in mice immunized with MIT-T-COVID vaccine significantly increased from 1.1% to 24.0% of total pulmonary nucleated cells prior to and at 7 days post infection (dpi), respectively, indicating dynamic recruitment of circulating specific T cells into the infected lungs. Mice immunized with MIT-T-COVID had 2.8 (2 dpi) and 3.3 (7 dpi) times more lung infiltrating CD8 + T cells than unimmunized mice. Mice immunized with MIT-T-COVID had 17.4 times more lung infiltrating CD4 + T cells than unimmunized mice (7 dpi). The undetectable specific antibody response in MIT-T-COVID-immunized mice demonstrates specific T cell responses alone can effectively attenuate the pathogenesis of SARS-CoV-2 infection. Our results suggest further study is merited for pan-variant T cell vaccines, including for individuals that cannot produce neutralizing antibodies or to help mitigate Long COVID., Competing Interests: DG and BC are founders of Think Therapeutics. PL and YT are employees of Acuitas Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Carter, Huang, Liu, Liang, Lin, Peng, McKay, Dimitrakakis, Hsu, Tat, Saenkham-Huntsinger, Chen, Kaseke, Gaiha, Xu, Griffiths, Tam, Tseng and Gifford.)

Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly- N -substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (<30 min) killing of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) at low micromolar concentrations without having apparent cytotoxic side effects in vivo . Additionally, these novel peptoids show excellent efficacy against biofilm formation and detachment for both MSSA and MRSA. In comparison, conventional antibiotics were unable to detach or prevent formation of biofilms. One cationic 12mer, Peptoid 1, shows great promise, as it could prevent formation of and detach biofilms at concentrations as low as 1.6 μM. The use of a bioluminescent S. aureus murine incision wound model demonstrated clearance of infection in peptoid-treated mice within 8 days, conveying another advantage these peptoids have over conventional antibiotics. These results provide clear evidence of the potential for antimicrobial peptoids for the treatment of S. aureus wound infections. IMPORTANCE Staphylococcus aureus resistance is a consistent problem with a large impact on the health care system. Infections with resistant S. aureus can cause serious adverse effects and can result in death. These antimicrobial peptoids show efficient killing of bacteria both as a biofilm and as free bacteria, often doing so in less than 30 min. As such, these antimicrobials have the potential to alleviate the burden that Staphylococcus infections have on the health care system and cause better outcomes for infected patients.

Urinary tract infection (UTI) is one of the most prevalent bacterial infections, particularly in women, children, and the elderly. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. Uropathogens are directly instilled in the urinary bladder, bypassing the lower urogenital tract, in the widely used murine model of UTI. We assessed whether vaginal inoculation of UPEC led to UTI and how stages of the estrous cycle would impact bacterial colonization in mice. Mice in proestrus, estrus, metestrus, and diestrus were identified by vaginal cytology and inoculated with UPEC in the vaginal tract. Mice were euthanized 1 day after infection, and bacterial loads in the urogenital tract, liver, and spleen were enumerated. Mice in estrus exhibited the highest and most consistent UPEC burdens in all organs, except the bladder. Vaginal inoculation resulted in bladder colonization in a UPEC strain-specific manner. In contrast, transurethral inoculation of UPEC led to bladder colonization. Importantly, inoculation by both routes led to vaginal and uterine colonization and concomitant systemic dissemination to the spleen and liver. The kinetics of bacterial colonization over 2 weeks following vaginal inoculation was comparable in the urogenital tract. Tissue sections revealed the induction of vaginitis and cystitis upon the vaginal instillation of UPEC. In summary, vaginal inoculation of UPEC in mice during estrus represents a novel approach to investigate infection of the kidneys and genital tract and systemic dissemination from the urogenital tract. Our findings suggest that estrogen primes the urogenital tract to create a conducive milieu for UPEC colonization.

Urinary tract infection (UTI) is one of the most common infectious conditions affecting people in the United States and around the world. Our knowledge of the host-pathogen interaction during UTI caused by Gram-positive bacterial uropathogens is limited compared to that for Gram-negative pathogens. Here, we investigated whether copper and the primary copper-containing protein, ceruloplasmin, are mobilized to urine during naturally occurring UTI caused by Gram-positive uropathogens in patients. Next, we probed the role of copper resistance in the fitness of methicillin-resistant Staphylococcus aureus (MRSA) during experimental UTI in a murine model. Our findings demonstrate that urinary copper and ceruloplasmin content are elevated during UTI caused by Enterococcus faecalis, S. aureus, S. epidermidis, and S. saprophyticus . MRSA strains successfully colonize the urinary tract of female CBA mice with selective induction of inflammation in the kidneys but not the bladder. MRSA mutants lacking CopL, a copper-binding cell surface lipoprotein, and the ACME genomic region containing copL , exhibit decreased fitness in the mouse urinary tract compared to parental strains. Copper sensitivity assays, cell-associated copper and iron content, and bioavailability of iron during copper stress demonstrate that homeostasis of copper and iron is interlinked in S. aureus. Importantly, relative fitness of the MRSA mutant lacking the ACME region is further decreased in mice that receive supplemental copper compared to the parental strain. In summary, copper is mobilized to the urinary tract during UTI caused by Gram-positive pathogens, and copper resistance is a fitness factor for MRSA during UTI. IMPORTANCE Urinary tract infection (UTI) is an extremely common infectious condition affecting people throughout the world. Increasing antibiotic resistance in pathogens causing UTI threatens our ability to continue to treat patients in the clinics. Better understanding of the host-pathogen interface is critical for development of novel interventional strategies. Here, we sought to elucidate the role of copper in host-Staphylococcus aureus interaction during UTI. Our results reveal that copper is mobilized to the urine as a host response in patients with UTI. Our findings from the murine model of UTI demonstrate that copper resistance is involved in the fitness of methicillin-resistant S. aureus (MRSA) during interaction with the host. We also establish a critical link between adaptation to copper stress and iron homeostasis in S. aureus.