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Staphylococcus aureus has been acquiring multiple drug resistance and has evolved into superbugs such as Methicillin/Vancomycin-resistant S. aureus (MRSA/VRSA) and, consequently, is a major cause of nosocomial and community infections associated with high morbidity and mortality for which no FDA-approved vaccines or biotherapeutics are available. Previous efforts targeting the surface-associated antigens have failed in clinical testing. Here, we generated hyperimmune products from sera in rabbits against six major S. aureus toxins targeted by an experimental vaccine (IBT-V02) and demonstrated significant efficacy for an anti-virulence passive immunization strategy. Extensive in vitro binding and neutralizing titers were analyzed against six extracellular toxins from individual animal sera. All IBT-V02 immunized animals elicited the maximum immune response upon the first boost dose against all pore-forming vaccine components, while for superantigen (SAgs) components of the vaccine, second and third doses of a boost were needed to reach a plateau in binding and toxin neutralizing titers. Importantly, both anti-staphylococcus hyperimmune products consisting of full-length IgG (IBT-V02-IgG) purified from the pooled sera and de-speciated F(ab')2 (IBT-V02-F(ab')2) retained the binding and neutralizing titers against IBT-V02 target toxins. F(ab')2 also exhibited cross-neutralization titers against three leukotoxins (HlgAB, HlgCB, and LukED) and four SAgs (SEC1, SED, SEK, and SEQ) which were not part of IBT-V02. F(ab')2 also neutralized toxins in bacterial culture supernatant from major clinical strains of S. aureus. In vivo efficacy data generated in bacteremia and pneumonia models using USA300 S. aureus strain demonstrated dose-dependent protection by F(ab')2. These efficacy data confirmed the staphylococcal toxins as viable targets and support the further development effort of hyperimmune products as a potential adjunctive therapy for emergency uses against life-threatening S. aureus infections.

The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.

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Gene deletion and the creation of pseudogenes have accompanied human adaptation in other Bordetella species7,8. In addition to the amino acid substitutions discussed above, 201 potential pseudogenes were created by indels and stop codons among the isolates (Supplementary Data 4). A gene ontology analysis demonstrated pseudogene enrichment in pathways relating to potassium ion transmembrane transport, proteolysis, cell adhesion, response to heat, and regulation of nitrogen compounds (Supplementary Fig. 12). Additionally, a gene annotated as thioredoxin reductase was both one of the most highly mutated genes in the isolate set and also a pseudogene in one lineage. These findings suggest that modification of the activity of the thioredoxin system, which funnels NADPH to reduce disulfide bonds in the cell, might have conferred a selective advantage under the conditions encountered in vivo38.

Hypermutators have been shown to occur in chronic infections and also on the shorter time scale of acute infection2,3,4,5,6. However, the importance of hypermutation in the context of zoonotic infection in a non-native host has not previously been studied in detail. In the case presented here, our findings argue that hypermutation resulted in a selective advantage through secondary adaptive mutations that were discovered and selected during the early course of clinical infection. These conclusions are independently supported by the observation that the DnaQ E9G hypermutators overtook the wild-type founder lineage in both blood and gastrointestinal compartments for a period of at least 12 months with clonal succession by multiple divergent hypermutating lineages, suggesting the superior in vivo fitness of these lineages. Several genes were repeatedly mutated in DnaQ E9G isolates and had an excess of non-synonymous mutations with dN/dS ratios >1. This suggests both that these genes were under positive selection and that the elevated mutation rates facilitated the discovery of adaptive mutations in DnaQ E9G. Though the hypermutator population dominated clinical cultures for a prolonged period during the clinical infection, the DnaQ E9G population eventually disappeared, and it is possible that this was a consequence of reduced viability resulting from the cumulative mutational load.

Glutathione and thioredoxin/thioredoxin reductase are critical components for maintaining cellular redox homeostasis and defense against oxidative stress. Targets involved in glutathione handling underwent mutation or inactivation during host adaptation, including genes annotated as a putative glutathione S-transferase (gstB_5), and glutathione hydrolase (ggt), among others. The linked thioredoxin/thioredoxin reductase system participates with glutathione in redox homeostasis and defense against oxidative attack by transferring reducing equivalents from NADPH to disulfide bonds in proteins. Remarkably, a gene annotated as a putative thioredoxin reductase (trxB_1) was both one of the most highly mutated genes with seven variants across the isolate set and also a pseudogene in one lineage. Together these findings in the glutathione and thioredoxin systems suggest specialized adaptation in the systems responding to oxidative damage, likely to be present from the mutational spectra observed. Whether a functioning thioredoxin system became maladaptive under the conditions of oxidative stress found in the host, perhaps by consuming excessive reduction potential, will need to be addressed by further experiments.

The third class of mutational targets included the B. hinzii homologs of the key histidine kinase transmembrane sensor proteins BvgS and RisS. The BvgS protein is a master regulator of virulence in B. pertussis, and signaling through this protein is required for B. pertussis colonization of the human respiratory tract37. RisS likewise plays a role as a master regulator of transcription in other Bordetella species, and additionally, the RisAS system interacts with the BvgAS system, though RisA may be the primary driver34. RisS is required for in vivo persistence in B. bronchiseptica33, but appears to be a pseudogene in human-adapted B. pertussis34. The roles and potential interaction of the BvgAS and RisAS systems in B. hinzii are not well studied, and evaluation of the consequences of the observed mutations would require further analysis.

In this approach, the reads for each lineage were aligned to the genome of the reconstructed ancestor using BWA 0.7.17 and soft clipping was allowed for a maximum of 10 bases using samclip v0.2. Duplicates were then removed and the.bam file was sorted using SAMtools v1.9. Variants were called using FreeBayes v1.3.1 in haploid mode and only accepting variants with a base quality of at least 30 and a mapping quality of at least 60. The variants were then filtered using BCFtools to keep calls with a QUAL of at least 100, a minimum coverage of 3, and a minimum alternative allele frequency of 0.66. The variants were then passed to Vt Normalize v0.5 to get consistent variant callings across the lineages. Finally, the ancestral variants present at the start of the experiment were removed to keep only those mutations that occurred during the experiment. Next, genomeCoverageBed from the BEDtools v2.72.1 package was used to identify which regions of the genome were at least covered by three reads in all the lineages, in order to compare the same regions of the genome across the different lineages and isolates. We then looked at the remaining variants that were common between different lineages. We were able to show that the majority of variants were shared by isolates derived from the same founding isolates, which was expected due to their relation in the first passage from the frozen stock. However, eight mutations were also identified that were shared across lineages descending from different founders and these were identified as true variants confirmed by inspection of mapping reads. To retain only the mutations that appeared independently during the last passage, we removed the mutations that appeared more than once across lineages in order to exclude the mutations that occurred during the first passage from frozen stock and before separation.

The authors can now show the robustness of their in silico data analysis by in vivo RT-qPCR measurements that experimentally confirm the downregulation of 5 hub genes. I believe the manuscript is now in better shape and can be considered for publication. 2351a5e196