Interkingdom interactions shape the fungal microbiome of mosquitoes.

BACKGROUND: The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (the mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). RESULTS: Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were < 1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize Aedes mosquitoes and support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. CONCLUSION: Collectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species.

Aedes aegypti gut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes.

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or the field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, many more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities is associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique for investigating host-microbe interactions in mosquitoes.

Evaluation of field resistance in field-collected mosquito Culex quinquefasciatus Say through quantification of ULV permethrin/PBO formulation in field bioassays.

BACKGROUND: Pyrethroids are among the most applied adulticides worldwide to control mosquito vectors for prevention of arboviral diseases transmission. However, pesticide resistance development in a mosquito population could lead to decreased control efficacy. While most studies investigate the resistant genotype (i.e. kdr, CYP450, etc.) as explanatory variables, few field efficacy studies have measured pesticide quantities deposited at different distances from the sprayer in association with observed mosquito mortality. The current study determined field delivered amounts of an applied ULV permethrin/PBO formulation (31% permethrin + 66% piperonyl butoxide) by GC/MS and estimated practical resistance ratios using caged mosquito females. RESULTS: For field samples, the extraction method recovered 78 ± 3.92-108 ± 8.97% of the permethrin/PBO formulation when utilizing the peaks of PBO from GC/MS to estimate the concentrations of adulticide deposited near the mosquito cages. The field bioassay showed that the spatial distribution of permethrin/PBO formulation was heterogeneous among three pseudo-replicates within the same distance. Within the quantifiable permethrin/PBO range of 15.7-51.4 ng/cm2 , field-collected mosquito mortalities started at 64% and linearly increased reaching 100% only in two areas, while all Sebring susceptible mosquitoes died. The field LC95 resistance ratio (RR) of F0 Cx. quinquefasciatus ranged from 2.65-3.51, falling within the 95% CI of RR95 estimated by laboratory vial assays. Tests with and without PBO indicated P450's enzymes contributed to field resistance. CONCLUSION: Results showed the suitability of the collection and quantification method to estimate the field resistance ratio at the applied pesticide rate. Pesticide quantification would also allow the association of the known frequencies of resistance mechanisms (e.g. kdr, CYP450) with field mortalities to estimate the resistance level conferred by such mechanisms. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Persistent variation in insecticide resistance intensity in container breeding Aedes (Diptera: Culicidae) co-collected in Houston, TX.

As observed in many locations worldwide, resistance to pyrethroids is common in Aedes aegypti (L.) in the southern United States and northern Mexico. Strong resistance in Aedes albopictus (Skuse) is less common and is not as well characterized. These 2 species have been undergoing range expansion and are sympatric in many locations including Houston, Texas. They are often collected from the same locations and lay eggs in the same larval habitats. In this study, we colonized both Ae. aegypti and Ae. albopictus from 4 locations in Houston and characterized insecticide resistance using permethrin as a model pyrethroid. We found differences in resistance intensity between the species at all 4 sites. Within the Ae. aegypti, resistance ratios ranged from 3.5- to 30.0-fold when compared to the ORL1952 laboratory susceptible strain. Expression of several P450s was higher than in the ORL1952 strain, but the pattern was similar between the field strains of Ae. aegypti. Higher resistance ratios did correlate with increasing percentages of the dilocus knockdown resistance (kdr) genotype. In contrast, Ae. albopictus from the 4 locations all had very low resistance ratios (<4-fold) when compared to the same laboratory susceptible strain. Five years later, we performed additional collections and characterization from the most resistant location to assess the temporal persistence of this difference in resistance between the species. The same pattern of high resistance in Ae. aegypti and low resistance in sympatric Ae. albopictus remained 5 yr later and this may have implications for operational efficacy.

Aedes aegypti gut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes.

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, far more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities are associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique.

Diagnosis of Acute Chagas Disease in a Belizean Child with Evidence of a Multiclonal Trypanosoma cruzi Infection.

In January 2020, we instituted acute febrile illness surveillance in 11 hospitals and clinics across Belize. Within 3 months, we diagnosed an acute case of Chagas disease by polymerase chain reaction in a 7-year-old child in the northern part of the country. Phylogenetic analyses of the parasite from the acute blood specimen revealed a multiclonal Trypanosoma cruzi infection, including parasites from the TcII (25.0% of haplotypes), TcIV (2.5% of haplotypes), and TcV (72.5% of haplotypes) discrete typing units. The family reported no history of travel, and three Triatoma species vectors were found within the home. The child's mother was seronegative for antibodies to T. cruzi, ruling out congenital transmission. Convalescent blood samples documented seroconversion and confirmed acute infection. The child was successfully treated with nifurtimox. This is the first known diagnosed case of acute Chagas infection in Belize, highlighting the need for further investigation and public health prevention measures.

Assessment of the Influence of Technology-Based Distracted Driving on Drivers' Infractions and Their Subsequent Impact on Traffic Accidents Severity.

Multitasking while driving negatively affects driving performance and threatens people's lives every day. Moreover, technology-based distractions are among the top driving distractions that are proven to divert the driver's attention away from the road and compromise their safety. This study employs recent data on road traffic accidents that occurred in Spain and uses a machine-learning algorithm to analyze, in the first place, the influence of technology-based distracted driving on drivers' infractions considering the gender and age of the drivers and the zone and the type of vehicle. It assesses, in the second place, the impact of drivers' infractions on the severity of traffic accidents. Findings show that (i) technology-based distractions are likely to increase the probability of committing aberrant infractions and speed infractions; (ii) technology-based distracted young drivers are more likely to speed and commit aberrant infractions; (iii) distracted motorcycles and squad riders are found more likely to speed; (iv) the probability of committing infractions by distracted drivers increases on streets and highways; and, finally, (v) drivers' infractions lead to serious injuries.

Microbial interactions in the mosquito gut determine Serratia colonization and blood-feeding propensity.

How microbe-microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that, Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however, one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behavior; Serratia exposure to Ae. aegypti disrupted their feeding behavior, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host-microbe interactions and provides evidence that microbial interactions influence mosquito behavior.

Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay.

We have developed a generalizable "smart molecular diagnostic" capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

Microbiome Interaction Networks and Community Structure From Laboratory-Reared and Field-Collected Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus Mosquito Vectors.

Microbial interactions are an underappreciated force in shaping insect microbiome communities. Although pairwise patterns of symbiont interactions have been identified, we have a poor understanding regarding the scale and the nature of co-occurrence and co-exclusion interactions within the microbiome. To characterize these patterns in mosquitoes, we sequenced the bacterial microbiome of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus caught in the field or reared in the laboratory and used these data to generate interaction networks. For collections, we used traps that attracted host-seeking or ovipositing female mosquitoes to determine how physiological state affects the microbiome under field conditions. Interestingly, we saw few differences in species richness or microbiome community structure in mosquitoes caught in either trap. Co-occurrence and co-exclusion analysis identified 116 pairwise interactions substantially increasing the list of bacterial interactions observed in mosquitoes. Networks generated from the microbiome of Ae. aegypti often included highly interconnected hub bacteria. There were several instances where co-occurring bacteria co-excluded a third taxa, suggesting the existence of tripartite relationships. Several associations were observed in multiple species or in field and laboratory-reared mosquitoes indicating these associations are robust and not influenced by environmental or host factors. To demonstrate that microbial interactions can influence colonization of the host, we administered symbionts to Ae. aegypti larvae that either possessed or lacked their resident microbiota. We found that the presence of resident microbiota can inhibit colonization of particular bacterial taxa. Our results highlight that microbial interactions in mosquitoes are complex and influence microbiome composition.

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone.

Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37°C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions.

Intestinal Myiasis Caused by Sarcophaga spp. in Cusco, Peru: A Case Report and Review of the Literature.

Myiasis is the infestation by dipterous fly larvae in humans and animals. The larvae can infect living or necrotic tissue involving the skin, nasopharynx, genitourinary, and gastrointestinal tracts. The accidental ingestion of eggs causes infection of the intestinal tract. We report a case of intestinal myiasis caused by Sarcophaga spp. larvae in a two-year-old child from Limatambo province in the Cusco region of Peru. Live larvae were identified incidentally in this child's stool sample during the study screening for Strongyloides stercoralis. The child did not have any constitutional or abdominal symptoms. The morphological examination of the specimen under magnification revealed Sarcophaga spp. larvae. We performed a literature review of publications reporting intestinal myiasis caused by Sarcophaga spp. and discussed key aspects of this infestation.

Global Transcriptome Analysis of Aedes aegypti Mosquitoes in Response to Zika Virus Infection.

Zika virus (ZIKV) of the Flaviviridae family is a recently emerged mosquito-borne virus that has been implicated in the surge of the number of microcephaly instances in South America. The recent rapid spread of the virus led to its declaration as a global health emergency by the World Health Organization. The virus is transmitted mainly by the mosquito Aedes aegypti, which is also the vector of dengue virus; however, little is known about the interactions of the virus with the mosquito vector. In this study, we investigated the transcriptome profiles of whole A. aegypti mosquitoes in response to ZIKV infection at 2, 7, and 14 days postinfection using transcriptome sequencing. Results showed changes in the abundance of a large number of transcripts at each time point following infection, with 18 transcripts commonly changed among the three time points. Gene ontology analysis revealed that most of the altered genes are involved in metabolic processes, cellular processes, and proteolysis. In addition, 486 long intergenic noncoding RNAs that were altered upon ZIKV infection were identified. Further, we found changes of a number of potential mRNA target genes correlating with those of altered host microRNAs. The outcomes provide a basic understanding of A. aegypti responses to ZIKV and help to determine host factors involved in replication or mosquito host antiviral response against the virus. IMPORTANCE Vector-borne viruses pose great risks to human health. Zika virus has recently emerged as a global threat, rapidly expanding its distribution. Understanding the interactions of the virus with mosquito vectors at the molecular level is vital for devising new approaches in inhibiting virus transmission. In this study, we embarked on analyzing the transcriptional response of Aedes aegypti mosquitoes to Zika virus infection. Results showed large changes in both coding and long noncoding RNAs. Analysis of these genes showed similarities with other flaviviruses, including dengue virus, which is transmitted by the same mosquito vector. The outcomes provide a global picture of changes in the mosquito vector in response to Zika virus infection.

Zika virus alters the microRNA expression profile and elicits an RNAi response in Aedes aegypti mosquitoes.

Zika virus (ZIKV), a flavivirus transmitted primarily by Aedes aegypti, has recently spread globally in an unprecedented fashion, yet we have a poor understanding of host-microbe interactions in this system. To gain insights into the interplay between ZIKV and the mosquito, we sequenced the small RNA profiles in ZIKV-infected and non-infected Ae. aegypti mosquitoes at 2, 7 and 14 days post-infection. ZIKA induced an RNAi response in the mosquito with virus-derived short interfering RNAs and PIWI-interacting RNAs dramatically increased in abundance post-infection. Further, we found 17 host microRNAs (miRNAs) that were modulated by ZIKV infection at all time points. Strikingly, many of these regulated miRNAs have been reported to have their expression altered by dengue and West Nile viruses, while the response was divergent from that induced by the alphavirus Chikungunya virus in mosquitoes. This suggests that conserved miRNA responses occur within mosquitoes in response to flavivirus infection. This study expands our understanding of ZIKV-vector interactions and provides potential avenues to be further investigated to target ZIKV in the mosquito host.

Microbial control of arthropod-borne disease.

Arthropods harbor a diverse array of microbes that profoundly influence many aspects of host biology, including vector competence. Additionally, symbionts can be engineered to produce molecules that inhibit pathogens. Due to their intimate association with the host, microbes have developed strategies that facilitate their transmission, either horizontally or vertically, to conspecifics. These attributes make microbes attractive agents for applied strategies to control arthropod-borne disease. Here we discuss the recent advances in microbial control approaches to reduce the burden of pathogens such as Zika, Dengue and Chikungunya viruses, and Trypanosome and Plasmodium parasites. We also highlight where further investigation is warranted.

Microbial control of arthropod-borne disease

Arthropods harbor a diverse array of microbes that profoundly influence many aspects of host biology, including vector competence. Additionally, symbionts can be engineered to produce molecules that inhibit pathogens. Due to their intimate association with the host, microbes have developed strategies that facilitate their transmission, either horizontally or vertically, to conspecifics. These attributes make microbes attractive agents for applied strategies to control arthropod-borne disease. Here we discuss the recent advances in microbial control approaches to reduce the burden of pathogens such as Zika, Dengue and Chikungunya viruses, and Trypanosome and Plasmodium parasites. We also highlight where further investigation is warranted.

A Multi-Serotype Approach Clarifies the Catabolite Control Protein A Regulon in the Major Human Pathogen Group A Streptococcus.

Catabolite control protein A (CcpA) is a highly conserved, master regulator of carbon source utilization in gram-positive bacteria, but the CcpA regulon remains ill-defined. In this study we aimed to clarify the CcpA regulon by determining the impact of CcpA-inactivation on the virulence and transcriptome of three distinct serotypes of the major human pathogen Group A Streptococcus (GAS). CcpA-inactivation significantly decreased GAS virulence in a broad array of animal challenge models consistent with the idea that CcpA is critical to gram-positive bacterial pathogenesis. Via comparative transcriptomics, we established that the GAS CcpA core regulon is enriched for highly conserved CcpA binding motifs (i.e. cre sites). Conversely, strain-specific differences in the CcpA transcriptome seems to consist primarily of affected secondary networks. Refinement of cre site composition via analysis of the core regulon facilitated development of a modified cre consensus that shows promise for improved prediction of CcpA targets in other medically relevant gram-positive pathogens.

Sequence type 1 group B Streptococcus, an emerging cause of invasive disease in adults, evolves by small genetic changes.

The molecular mechanisms underlying pathogen emergence in humans is a critical but poorly understood area of microbiologic investigation. Serotype V group B Streptococcus (GBS) was first isolated from humans in 1975, and rates of invasive serotype V GBS disease significantly increased starting in the early 1990s. We found that 210 of 229 serotype V GBS strains (92%) isolated from the bloodstream of nonpregnant adults in the United States and Canada between 1992 and 2013 were multilocus sequence type (ST) 1. Elucidation of the complete genome of a 1992 ST-1 strain revealed that this strain had the highest homology with a GBS strain causing cow mastitis and that the 1992 ST-1 strain differed from serotype V strains isolated in the late 1970s by acquisition of cell surface proteins and antimicrobial resistance determinants. Whole-genome comparison of 202 invasive ST-1 strains detected significant recombination in only eight strains. The remaining 194 strains differed by an average of 97 SNPs. Phylogenetic analysis revealed a temporally dependent mode of genetic diversification consistent with the emergence in the 1990s of ST-1 GBS as major agents of human disease. Thirty-one loci were identified as being under positive selective pressure, and mutations at loci encoding polysaccharide capsule production proteins, regulators of pilus expression, and two-component gene regulatory systems were shown to affect the bacterial phenotype. These data reveal that phenotypic diversity among ST-1 GBS is mainly driven by small genetic changes rather than extensive recombination, thereby extending knowledge into how pathogens adapt to humans.

Characterization of the effect of the histidine kinase CovS on response regulator phosphorylation in group A Streptococcus.

Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections.

Dual-site phosphorylation of the control of virulence regulator impacts group a streptococcal global gene expression and pathogenesis.

Phosphorylation relays are a major mechanism by which bacteria alter transcription in response to environmental signals, but understanding of the functional consequences of bacterial response regulator phosphorylation is limited. We sought to characterize how phosphorylation of the control of virulence regulator (CovR) protein from the major human pathogen group A Streptococcus (GAS) influences GAS global gene expression and pathogenesis. CovR mainly serves to repress GAS virulence factor-encoding genes and has been shown to homodimerize following phosphorylation on aspartate-53 (D53) in vitro. We discovered that CovR is phosphorylated in vivo and that such phosphorylation is partially heat-stable, suggesting additional phosphorylation at non-aspartate residues. Using mass spectroscopy along with targeted mutagenesis, we identified threonine-65 (T65) as an additional CovR phosphorylation site under control of the serine/threonine kinase (Stk). Phosphorylation on T65, as mimicked by the recombinant CovR T65E variant, abolished in vitro CovR D53 phosphorylation. Similarly, isoallelic GAS strains that were either unable to be phosphorylated at D53 (CovR-D53A) or had functional constitutive phosphorylation at T65 (CovR-T65E) had essentially an identical gene repression profile to each other and to a CovR-inactivated strain. However, the CovR-D53A and CovR-T65E isoallelic strains retained the ability to positively influence gene expression that was abolished in the CovR-inactivated strain. Consistent with these observations, the CovR-D53A and CovR-T65E strains were hypervirulent compared to the CovR-inactivated strain in a mouse model of invasive GAS disease. Surprisingly, an isoalleic strain unable to be phosphorylated at CovR T65 (CovR-T65A) was hypervirulent compared to the wild-type strain, as auto-regulation of covR gene expression resulted in lower covR gene transcript and CovR protein levels in the CovR-T65A strain. Taken together, these data establish that CovR is phosphorylated in vivo and elucidate how the complex interplay between CovR D53 activating phosphorylation, T65 inhibiting phosphorylation, and auto-regulation impacts streptococcal host-pathogen interaction.