Expressing the pro-apoptotic Reaper protein via insertion into the structural open reading frame of Sindbis virus reduces the ability to infect Aedes aegypti mosquitoes

Arboviruses continue to threaten a significant portion of the human population, and a better understanding is needed of the determinants of successful arbovirus infection of arthropod vectors. Avoiding apoptosis has been shown to be one such determinant. Previous work showed that a Sindbis virus (SINV) construct called MRE/rpr that expresses the pro-apoptotic protein Reaper via a duplicated subgenomic promoter had a reduced ability to orally infect Aedes aegypti mosquitoes at 3 days post-blood meal (PBM), but this difference diminished over time as virus variants containing deletions in the inserted reaper gene rapidly predominated. The goal of this study was to generate a SINV construct that more stably expressed Reaper, in order to further clarify the effect of midgut apoptosis on disseminated infection in Ae. aegypti. We did this by inserting reaper as an in-frame fusion into the structural open reading frame (ORF) of SINV. This construct, MRE/rprORF, successfully expressed Reaper, replicated similarly to MRE/rpr in cell lines, and induced apoptosis in cultured cells and in mosquito midgut tissue. Mosquitoes that fed on blood containing MRE/rprORF developed less midgut and disseminated infection when compared to MRE/rpr or a control virus up to at least 7 days PBM, when less than 50% of mosquitoes that ingested MRE/rprORF had detectable disseminated infection, compared with around 80% or more of mosquitoes fed with MRE/rpr or control virus. However, virus titer in mosquitoes infected with MRE/rprORF was not significantly different from control virus, suggesting that induction of apoptosis by expression of Reaper by this method can reduce infection prevalence, but if infection is established then apoptosis induced by this method has limited ability to continue to suppress replication.

260. The Unfortunate Consequence of Immunosuppression in a Renal Transplant Patient

Background Nocardia is a slow-growing aerobic-actinomycete that belongs to the family Nocardiaceae. Major predisposing factors include corticosteroid use, organ transplantation, low CD4 count, and hematologic malignancies. The most commonly affected organs are lungs, mainly via inhalation; however, the most common extrapulmonary site is central nervous system. Methods Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-ToF) or 16srRNA sequencing are more reliable methodologies for accurate identification of Nocardia to the species level. To our knowledge, our patient represented the first U.S. case of N. bejingensis opportunistic disseminated infection in a renal transplant patient although similar cases have been previously reported outside the U.S. GMS stain Gram Stain of Nocardia Results We present a 31-year-old Caucasian male status post renal transplant four years ago on immunosuppressants with left arm myoclonic jerks. In addition, there was an associated unilateral left frontal headache of four to five day duration. His chest CT revealed consolidative process in the right lower lobe and pleural effusion. MRI of the brain revealed multiple ring-enhancing lesions. Patient underwent left frontal craniotomy with resection and a complete evacuation of brain abscess. His brain abscess and pleural fluid cultures revealed Gram positive rods, which were subsequently identified as Nocardia beijingensis by MALDI-TOF and confirmed by 16srRNA sequencing. He was treated with intravenous imipenem & trimethoprim – sulfamethoxazole with subsequent clinical improvement. MRI Brain w/ contrast Head CT s/p left frontal craniotomy with resection & evacuation of abscess Chest CT Conclusion Different Nocardia species have a wide geographic distribution with varying pathogenic traits, and antimicrobial susceptibility. Hence, the identification of the specific species of Nocardia is crucial to provide a proficient level of patient care. Nocardia bejingensis is a newly discovered species of Nocardia that was first isolated in 2001 in China. Only six cases of N. beijingensis affecting CNS have been reported up to date in the United States. It is unclear of the geographic distribution and variable antimicrobial susceptibility of Nocardia bejingensis but we can confirm the first reported case of an opportunistic disseminated infection in a renal transplant patient in the United States. Agar Disclosures All Authors: No reported disclosures

830. Central Nervous System Involvement in Disseminated Mycobacterium avium Complex Infection in Patients with Newly Diagnosed HIV

Background Disseminated Mycobacterium avium complex (MAC) infection occurs in 20-40% of patients with < 50 CD4/mm3. Data describing central nervous MAC involvement (CNS-MAC) in disseminated infection is scarce. Methods We conducted a retrospective case series in the outpatient infectious diseases clinic in the hospital “Dr. Manuel Gea Gonzales” in Mexico City. We reviewed all records from October 2020 to May 2021 and identified all culture proven MAC infections. Results We found 7 cases of MAC, with disseminated infection (positive bone marrow cultures) with 3 out of those 7 meeting our definition for CNS-MAC (positive cerebrospinal fluid culture). All cases of CNS-MAC infection occurred in patients with < 50 CD4/mm3 and recent HIV diagnosis (1-4 months) that were referred to our institution with consumptive syndrome and fevers. All patients were receiving antiretroviral treatment (ART) with BIC/FTC/TAF and initiated ART in less than 1 month since HIV diagnosis. Opportunistic infections were ruled-out at the moment of CNS-MAC diagnosis (criptococcal meningitis, cytomegalovirus retinitis, tuberculosis and histoplasmosis). All patients exhibited non-specific neurologic symptoms at arrival (headache and bradipsiquia) mixed with more severe symptoms (one case of ataxia, one case of vertigo, one case of III nerve palsy). All patients were treated with Clarithromycin/Levofloxacin/Ethambutol. Two patients achieved symptom remission and 1 patient was lost to follow-up. Of important note, all CSF analysis and CNS imaging studies carried-out were normal. No MAC bacilli were identified with direct Ziel-Neelsen staining of CSF. Conclusion We found a high proportion of CNS-MAC in patients with disseminated MAC infection (42.8%) during the study period. All patients presented CNS symptoms and normal CSF characteristics. In our setting, patients with suspected disseminated MAC infection CD4 counts < 50 cells/mm3 might represent a specific population that could benefit from routine targeted diagnostic test at presentation in order to establish CNS involvement. Disclosures All Authors: No reported disclosures

Copper intoxication in group B Streptococcus triggers transcriptional activation of the cop operon that contributes to enhanced virulence during acute infection

Bacteria can utilize Copper (Cu) as a trace element to support cellular processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B streptococcus (GBS), and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of GBS mutants deficient in either the copA Cu exporter, or the copY repressor, were severely compromised in Cu-stress conditions. GBS survival of Cu stress reflected a mechanism of CopY de-repression of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA-sequencing revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication, via regulation by the Cu-sensing transcriptional repressor, copY . Cu stress responses in GBS reflect a transcriptional signature that heightens virulence and represents an important part of the bacteria’s ability to survive in different environments. Importance Understanding how bacteria manage cellular levels of metal ions, such as copper, helps to explain how microbial cells can survive in different stressful environments. We show how the opportunistic pathogen group B Streptococcus (GBS) achieves homeostasis of intracellular copper through the activities of the genes that comprise the cop operon, and describe how this helps GBS survive in stressful environments, including in the mammalian host during systemic disseminated infection.

Evaluating the vector competence of Aedes simpsoni sl from Kenyan coast for Ngari and Bunyamwera viruses

Background Bunyamwera(BUNV) and Ngari (NGIV) viruses are arboviruses of medical importance globally, the viruses are endemic in Africa, Aedes(Ae) aegypti and Anopheles(An) gambiae mosquitoes are currently competent vectors for BUNV and NGIV respectively. Both viruses have been isolated from humans and mosquitoes in various ecologies of Kenya. Understanding the risk patterns and spread of the viruses necessitate studies of vector competence in local vector population of Ae. simpsoni sl which is abundant in the coastal region. This study sought to assess the ability of Ae. Simpsoni sl mosquitoes abundant at the Coast of Kenya to transmit these viruses in experimental laboratory experiments. Methods Field collected larvae/pupae of Ae. Simpsoni sl mosquitoes from Rabai, Kilifi County, were reared to adults, the first filial generation (F0) females’ mosquitoes were orally exposed to infectious blood meal with isolates of the viruses using the hemotek membrane feeder. The exposed mosquitoes were incubated under insectary conditions and sampled on day 7, 14 and 21days post infection to determine susceptibility to the virus infection using plaque assay. Results A total of 379 (Bunyamwera virus 255 and Ngari virus 124) Ae. simpsoni sl were orally exposed to infectious blood meal. Overall, the infection rate (IR) for BUNV and NGIV were 2.7 and 0.9% respectively. Dissemination occurred in 5 out 7 mosquitoes with mid-gut infection for Bunyamwera virus and 1 out of 2 mosquitoes with mid-gut infection for Ngari virus. Further, the transmission was observed in 1 out of 5 mosquitoes that had disseminated infection and no transmission was observed for Ngari virus in all days post infection (dpi). Conclusion Our study shows that Ae. simpsoni sl. is a laboratory competent vector for Bunyamwera virus since it was able to transmit the virus through capillary feeding while NGIV infection was restricted to midgut infection and disseminated infection, these finding adds information on the epidemiology of the viruses and vector control plan.

The infectivity gene bbk13 is important for multiple phases of the Borrelia burgdorferi enzootic cycle

Lyme disease is a multi-stage inflammatory disease caused by the spirochete Borrelia burgdorferi transmitted through the bite of an infected Ixodes scapularis tick. We previously discovered a B. burgdorferi infectivity gene, bbk13 , that facilitates mammalian infection by promoting spirochete population expansion in the skin inoculation site. Initial characterization of bbk13 was carried out using an intradermal needle inoculation model of mouse infection, which does not capture the complex interplay of the pathogen-vector-host triad of natural transmission. Herein, we aimed to understand the role of bbk13 in the enzootic cycle of B. burgdorferi . B. burgdorferi lacking bbk13 were unable to be acquired by naive larvae fed on needle inoculated mice. Using a capsule-feeding approach to restrict tick feeding activity to a defined skin site, we determined that delivery by tick bite alleviated the population expansion defect in the skin observed after needle inoculation of Δ bbk13 B. burgdorferi . Despite overcoming the early barrier in the skin, Δ bbk13 B. burgdorferi remained attenuated for distal tissue colonization after tick transmission. Disseminated infection of Δ bbk13 B. burgdorferi was improved in needle inoculated immunocompromised mice. Together, we established that bbk13 is crucial to the maintenance of B. burgdorferi in the enzootic cycle and that bbk13 is necessary beyond early infection in the skin, likely contributing to host immune evasion. Moreover, our data highlight the critical interplay between the pathogen, vector, and host as well as the distinct molecular genetic requirements for B. burgdorferi to survive at the pathogen-vector-host interface and to achieve productive disseminated infection.