Aerosolized aqueous dust extracts collected near a drying lake trigger acute neutrophilic pulmonary inflammation reminiscent of microbial innate immune ligands.

BACKGROUND: A high incidence of asthma is prevalent among residents near the Salton Sea, a large inland terminal lake in southern California. This arid region has high levels of ambient particulate matter (PM); yet while high PM levels are often associated with asthma in many environments, it is possible that the rapidly retreating lake, and exposed playa or lakebed, may contribute components with a specific role in promoting asthma symptoms. OBJECTIVES: Our hypothesis is that asthma may be higher in residents closest to the Salton Sea due to chronic exposures to playa dust. Playa emissions may be concentrating dissolved material from the lake, with microbial components capable of inducing pulmonary innate immune responses. To test this hypothesis, we used a mouse model of aerosol exposures to assess the effects of playa dust. METHODS: From dust collected around the Salton Sea region, aqueous extracts were used to generate aerosols, which were injected into an environmental chamber for mouse exposure studies. We compared the effects of exposure to Salton Sea aerosols, as well as to known immunostimulatory reference materials. Acute 48-h and chronic 7-day exposures were compared, with lungs analyzed for inflammatory cell recruitment and gene expression. RESULTS: Dust from sites nearest to the Salton Sea triggered lung neutrophil inflammation that was stronger at 48-h but reduced at 7-days. This acute inflammatory profile and kinetics resembled the response to innate immune ligands LTA and LPS while distinct from the classic allergic response to Alternaria. CONCLUSION: Lung inflammatory responses to Salton Sea dusts are similar to acute innate immune responses, raising the possibility that microbial components are entrained in the dust, promoting inflammation. This effect highlights the health risks at drying terminal lakes from inflammatory components in dust emissions from exposed lakebed.

Salton Sea aerosol exposure in mice induces a pulmonary response distinct from allergic inflammation.

In communities surrounding the Salton Sea, high rates of asthma are associated with high aerosol dust levels. However, the Salton Sea itself may play an additional role in pulmonary health. Therefore, to investigate a potential role of the Salton Sea on pulmonary health, we exposed mice to aerosolized Salton Sea water for 7 days and assessed tissue responses, including cellular infiltration and gene expression changes. For reference, mice were also exposed to aerosolized fungal allergen (Alternaria sp.) and Pacific Ocean aerosols. Exposure to aerosolized Alternaria sp. induced dramatic allergic inflammation, including neutrophil and eosinophil recruitment to the bronchoalveolar lavage fluid (BALF) and lung tissue. By contrast, Salton Sea "spray" induced only B cell recruitment to the lung tissue without increased inflammatory cell numbers in BALF. However, there were consistent gene expression changes suggestive of an inflammatory response. The response to the Salton Sea spray was notably distinct from the response to Pacific Ocean water, which induced some B cell recruitment but without an inflammatory gene expression profile. Our studies suggest that soluble components in Salton Sea water promote induction of a unique inflammation-associated response, though any relationship to asthma remains to be explored.

Agrobacterium tumefaciens Growth Pole Ring Protein: C Terminus and Internal Apolipoprotein Homologous Domains Are Essential for Function and Subcellular Localization.

The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium-specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth.IMPORTANCE Bacterial growth and division are extensively studied in model systems (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode-polar growth-is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae, in which many species, including Agrobacterium tumefaciens, exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.

Mapping Neutralizing Antibody Epitope Specificities to an HIV Env Trimer in Immunized and in Infected Rhesus Macaques.

BG505 SOSIP is a well-characterized near-native recombinant HIV Envelope (Env) trimer that holds promise as part of a sequential HIV immunogen regimen to induce broadly neutralizing antibodies (bnAbs). Rhesus macaques are considered the most appropriate pre-clinical animal model for monitoring antibody (Ab) responses. Accordingly, we report here the isolation of 45 BG505 autologous neutralizing antibodies (nAbs) with multiple specificities from SOSIP-immunized and BG505 SHIV-infected rhesus macaques. We associate the most potent neutralization with two epitopes: the C3/V5 and V1/V3 regions. We show that all of the nAbs bind in close proximity to known bnAb epitopes and might therefore sterically hinder elicitation of bnAbs. We also identify a "public clonotype" that targets the immunodominant C3/V5 nAb epitope, which suggests that common antibody rearrangements might help determine humoral responses to Env immunogens. The results highlight important considerations for vaccine design in anticipation of results of the BG505 SOSIP trimer in clinical trials.

Staphylococcus aureus Fibronectin Binding Protein A Mediates Biofilm Development and Infection.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.

Rapid and Focused Maturation of a VRC01-Class HIV Broadly Neutralizing Antibody Lineage Involves Both Binding and Accommodation of the N276-Glycan.

The VH1-2 restricted VRC01-class of antibodies targeting the HIV envelope CD4 binding site are a major focus of HIV vaccine strategies. However, a detailed analysis of VRC01-class antibody development has been limited by the rare nature of these responses during natural infection and the lack of longitudinal sampling of such responses. To inform vaccine strategies, we mapped the development of a VRC01-class antibody lineage (PCIN63) in the subtype C infected IAVI Protocol C neutralizer PC063. PCIN63 monoclonal antibodies had the hallmark VRC01-class features and demonstrated neutralization breadth similar to the prototype VRC01 antibody, but were 2- to 3-fold less mutated. Maturation occurred rapidly within ∼24 months of emergence of the lineage and somatic hypermutations accumulated at key contact residues. This longitudinal study of broadly neutralizing VRC01-class antibody lineage reveals early binding to the N276-glycan during affinity maturation, which may have implications for vaccine design.