Single-domain antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer surface protein A (OspA).

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.

Structure of a transmission blocking antibody in complex with Outer surface protein A from the Lyme disease spirochete, Borreliella burgdorferi.

319-44 is a human monoclonal antibody capable of passively protecting mice against tick-mediated infection with Borreliella burgdorferi, the bacterial genospecies responsible for Lyme disease in North America. In vitro, 319-44 has complement-dependent borreliacidal activity and spirochete agglutinating properties. Here, we report the 2.2 Å-resolution crystal structure of 319-44 Fab fragments in complex with Outer surface protein A (OspA), the ~30 kDa lipoprotein that was the basis of the first-generation Lyme disease vaccine approved in the United States. The 319-44 epitope is focused on OspA ß-strands 19, 20, and 21, and the loops between ß-strands 16-17, 18-19, and 20-21. Contact with loop 20-21 explains competition with LA-2, the murine monoclonal antibody used to estimate serum borreliacidal activities in the first-generation Lyme disease vaccine clinical trials. A high-resolution B-cell epitope map of OspA will accelerate structure-based design of second generation OspA-based vaccines.

Transcytosis of IgA Attenuates Salmonella Invasion in Human Enteroids and Intestinal Organoids.

Secretory IgA (SIgA) is the most abundant antibody type in intestinal secretions where it contributes to safeguarding the epithelium from invasive pathogens like the Gram-negative bacterium, Salmonella enterica serovar Typhimurium (STm). For example, we recently reported that passive oral administration of the recombinant monoclonal SIgA antibody, Sal4, to mice promotes STm agglutination in the intestinal lumen and restricts bacterial invasion of Peyer's patch tissues. In this report, we sought to recapitulate Sal4-mediated protection against STm in human Enteroids and human intestinal organoids (HIOs) as models to decipher the molecular mechanisms by which antibodies function in mucosal immunity in the human gastrointestinal tract. We confirm that Enteroids and HIO-derived monolayers are permissive to STm infection, dependent on HilD, the master transcriptional regulator of the SPI-I type three secretion system (T3SS). Stimulation of M-like cells in both Enteroids and HIOs by the addition of RANKL further enhanced STm invasion. The apical addition of Sal4 mouse IgA, as well as recombinant human Sal4 dimeric IgA (dIgA) and SIgA resulted a dose-dependent reduction in bacterial invasion. Moreover, basolateral application of Sal4 dIgA to Enteroid and HIO monolayers gave rise to SIgA in the apical compartment via a pathway dependent on expression of the polymeric immunoglobulin receptor (pIgR). The resulting Sal4 SIgA was sufficient to reduce STm invasion of Enteroid and HIO epithelial cell monolayers by ~20-fold. Recombinant Sal4 IgG was also transported in the Enteroid and HIOs, but to a lesser degree and via a pathway dependent on the neonatal Fc receptor (FCGRT). The models described lay the foundation for future studies into detailed mechanisms of IgA and IgG protection against STm and other pathogens.

Creatine utilization as a sole nitrogen source in Pseudomonas putida KT2440 is transcriptionally regulated by CahR.

Glutamine amidotransferase-1 domain-containing AraC-family transcriptional regulators (GATRs) are present in the genomes of many bacteria, including all Pseudomonas species. The involvement of several characterized GATRs in amine-containing compound metabolism has been determined, but the full scope of GATR ligands and regulatory networks are still unknown. Here, we characterize Pseudomonas putida's detection of the animal-derived amine compound creatine, a compound particularly enriched in muscle and ciliated cells by a creatine-specific GATR, PP_3665, here named CahR (Creatine amidohydrolase Regulator). cahR is necessary for transcription of the gene encoding creatinase (PP_3667/creA) in the presence of creatine and is critical for P. putida's ability to utilize creatine as a sole source of nitrogen. The CahR/creatine regulon is small, and an electrophoretic mobility shift assay demonstrates strong and specific CahR binding only at the creA promoter, supporting the conclusion that much of the regulon is dependent on downstream metabolites. Phylogenetic analysis of creA orthologues associated with cahR orthologues highlights a strain distribution and organization supporting probable horizontal gene transfer, particularly evident within the genus Acinetobacter. This study identifies and characterizes the GATR that transcriptionally controls P. putida's metabolism of creatine, broadening the scope of known GATR ligands and suggesting GATR diversification during evolution of metabolism for aliphatic nitrogen compounds.

Characterizing species interactions that contribute to biofilm formation in a multispecies model of a potable water bacterial community.

Microbial biofilms are ubiquitous in drinking water systems, yet our understanding of drinking water biofilms lags behind our understanding of those in other environments. Here, a six-member model bacterial community was used to identify the interactions and individual contributions of each species to community biofilm formation. These bacteria were isolated from the International Space Station potable water system and include Cupriavidus metallidurans, Chryseobacterium gleum, Ralstonia insidiosa, Ralstonia pickettii, Methylorubrum (Methylobacterium) populi and Sphingomonas paucimobilis, but all six species are common members of terrestrial potable water systems. Using reconstituted assemblages, from pairs to all 6 members, community biofilm formation was observed to be robust to the absence of any single species and only removal of the C. gleum/S. paucimobilis pair, out of all 15 possible 2-species subtractions, led to loss of community biofilm formation. In conjunction with these findings, dual-species biofilm formation assays supported the view that the contribution of C. gleum to community biofilm formation was dependent on synergistic biofilm formation with either R. insidiosa or C. metallidurans. These data support a model of multiple, partially redundant species interactions to generate robustness in biofilm formation. A bacteriophage and multiple predatory bacteria were used to test the resilience of the community to the removal of individual members in situ, but the combination of precise and substantial depletion of a single target species was not achievable. We propose that this assemblage can be used as a tractable model to understand the molecular bases of the interactions described here and to decipher other functions of drinking water biofilms.

Stenotrophomonas maltophilia Differential Gene Expression in Synthetic Cystic Fibrosis Sputum Reveals Shared and Cystic Fibrosis Strain-Specific Responses to the Sputum Environment.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can infect the lungs of people with cystic fibrosis (CF). The highly viscous mucus in the CF lung, expectorated as sputum, serves as the primary nutrient source for microbes colonizing this site and induces virulence-associated phenotypes and gene expression in several CF pathogens. Here, we characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic CF sputum medium (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect the metabolic pathways used by S. maltophilia in sputum, as well as altered stress responses. The latter correlated with increased resistance to peroxide exposure after pregrowth in SCFM2 for two of the strains. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates to that of the acute infection strain, S. maltophilia K279a, allowing us to identify CF isolate-specific signatures in differential gene expression. The expression of genes from the accessory genomes was also differentially altered in response to SCFM2. Finally, a number of biofilm-associated genes were differentially induced in SCFM2, particularly in K279a, which corresponded to increased aggregation and biofilm formation in this strain relative to both CF strains. Collectively, this work details the response of S. maltophilia to an environment that mimics important aspects of the CF lung, identifying potential survival strategies and metabolic pathways used by S. maltophilia during infections.IMPORTANCEStenotrophomonas maltophilia is an important infecting bacterium in the airways of people with cystic fibrosis (CF). However, compared to the other CF pathogens, S. maltophilia has been relatively understudied. The significance of our research is to provide insight into the global transcriptomic changes of S. maltophilia in response to a medium that was designed to mimic important aspects of the CF lung. This study elucidates the overall metabolic changes that occur when S. maltophilia encounters the CF lung and generates a road map of candidate genes to test using in vitro and in vivo models of CF.

Pulmonary Surfactant Promotes Virulence Gene Expression and Biofilm Formation in Klebsiella pneumoniae.

The interactions between Klebsiella pneumoniae and the host environment at the site of infection are largely unknown. Pulmonary surfactant serves as an initial point of contact for inhaled bacteria entering the lung and is thought to contain molecular cues that aid colonization and pathogenesis. To gain insight into this ecological transition, we characterized the transcriptional response of K. pneumoniae MGH 78578 to purified pulmonary surfactant. This work revealed changes within the K. pneumoniae transcriptome that likely contribute to host colonization, adaptation, and virulence in vivo Notable transcripts expressed under these conditions include genes involved in capsule synthesis, lipopolysaccharide modification, antibiotic resistance, biofilm formation, and metabolism. In addition, we tested the contributions of other surfactant-induced transcripts to K. pneumoniae survival using engineered isogenic KPPR1 deletion strains in a murine model of acute pneumonia. In these infection studies, we identified the MdtJI polyamine efflux pump and the ProU glycine betaine ABC transporter to be significant mediators of K. pneumoniae survival within the lung and confirmed previous evidence for the importance of de novo leucine synthesis to bacterial survival during infection. Finally, we determined that pulmonary surfactant promoted type 3 fimbria-mediated biofilm formation in K. pneumoniae and identified two surfactant constituents, phosphatidylcholine and cholesterol, that drive this response. This study provides novel insight into the interactions occurring between K. pneumoniae and the host at an important infection site and demonstrates the utility of purified lung surfactant preparations for dissecting host-lung pathogen interactions in vitro.

Differential requirements for processing and transport of short-chain versus long-chain O-acylcarnitines in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa can metabolize carnitine and O-acylcarnitines, which are abundant in host muscle and other tissues. Acylcarnitines are metabolized to carnitine and a fatty acid. The liberated carnitine and its catabolic product, glycine betaine, can be used as osmoprotectants, to induce the secreted phospholipase C PlcH, and as sole carbon, nitrogen and energy sources. P. aeruginosa is incapable of de novo synthesis of carnitine and acylcarnitines, therefore they must be imported from an exogenous source. In this study, we present the first characterization of bacterial acylcarnitine transport. Short-chain acylcarnitines are imported by the ABC transporter CaiX-CbcWV. Medium- and long-chain acylcarnitines (MCACs and LCACs) are hydrolysed extracytoplasmically and the free carnitine is transported primarily through CaiX-CbcWV. These findings suggest that the periplasmic protein CaiX has a binding pocket that permits short acyl chains on its carnitine ligand and that there are one or more secreted hydrolases that cleave MCACs and LCACs. To identify the secreted hydrolase(s), we used a saturating genetic screen and transcriptomics followed by phenotypic analyses, but neither led to identification of a contributing hydrolase, supporting but not conclusively demonstrating redundancy for this activity.

Transcriptional Responses of Pseudomonas aeruginosa to Potable Water and Freshwater.

Many Pseudomonas aeruginosa infections are derived from residential, recreational, or surface water sources; thus, these environments represent an important preinfection niche. To better understand P. aeruginosa biology in these environments, we quantified transcriptional changes by microarray after exposure to diluted LB, diluted R2B, potable tap water, and freshwater from a eutrophic pond. Quantitative reverse transcription-PCR (qRT-PCR) confirmed the conservation of these responses in other water sources, and competition experiments were used to test the importance of three implicated metabolic pathways. The global transcriptional responses in potable water and freshwater showed strong induction of genes involved in metabolism of the head groups and acyl tails of phospholipids, as well as nucleotide metabolism, with commensurate decreased transcript expression of genes encoding their synthetic pathways. These data suggest that phospholipids and nucleotides are part of the nutritional milieu of these two environments. A unique response in municipal-delivered potable water was to the metals in the piping system, particularly copper. To identify potential nutrient sources used by P. aeruginosa in these environments, we used competition assays between the wild-type and deletion mutant strains in three pathways induced under these conditions. For phospholipid head-group metabolism, ethanolamine utilization (eutB) was important for competition in potable water, while choline oxidation (betBA) was important for competition in freshwater. Nucleotide utilization, particularly pyrimidine metabolism (dht), showed a trend toward importance in freshwater but was not statistically significant. These findings provide new insights into the P. aeruginosa response to potable water and freshwater and led to the identification of potentially important nutrient sources in these environments.IMPORTANCE Much of our knowledge about Pseudomonas aeruginosa comes from the infection niche, and much less is known about its lifestyle in the environment. P. aeruginosa is an adaptable bacterium capable of growing in many environments but is particularly common in potable water systems and freshwater. We used the transcriptional responses of P. aeruginosa to these environments to identify important nutrient sources specific to either of these two environments. Additionally, these environments could provide experimental situations to understand gene function for the large number of transcripts with unknown functions induced under these conditions.

Sarcosine Catabolism in Pseudomonas aeruginosa Is Transcriptionally Regulated by SouR.

UNLABELLED: Sarcosine (N-methylglycine) is present in many environments inhabited by pseudomonads and is likely most often encountered as an intermediate in the metabolism of choline, carnitine, creatine, and glyphosate. While the enzymology of sarcosine metabolism has been relatively well studied in bacteria, the regulatory mechanisms governing catabolism have remained largely unknown. We previously determined that the sarcosine-catabolic (sox) operon of Pseudomonas aeruginosa is induced by the AraC family regulator GbdR in response to glycine betaine and dimethylglycine. However, induction of these genes was still observed in response to sarcosine in a gbdR deletion mutant, indicating that an independent sarcosine-responsive transcription factor also acted at this locus. Our goal in this study was to identify and characterize this regulator. Using a transposon-based genetic screen, we identified PA4184, or SouR (sarcosine oxidation and utilization regulator), as the sarcosine-responsive regulator of the sox operon, with tight induction specificity for sarcosine. The souR gene is required for appreciable growth on sarcosine as a carbon and nitrogen source. We also characterized the transcriptome response to sarcosine governed by SouR using microarray analyses and performed electrophoretic mobility shift assays to identify promoters directly regulated by the transcription factor. Finally, we characterized PA3630, or GfnR (glutathione-dependent formaldehyde neutralization regulator), as the regulator of the glutathione-dependent formaldehyde detoxification system in P. aeruginosa that is expressed in response to formaldehyde released during the catabolism of sarcosine. This study expands our understanding of sarcosine metabolic regulation in bacteria through the identification and characterization of the first known sarcosine-responsive transcriptional regulator. IMPORTANCE: The Pseudomonas aeruginosa genome encodes many diverse metabolic pathways, yet the specific transcription regulators controlling their expression remain mostly unknown. Here, we used a genetic screen to identify the sarcosine-specific regulator of the sarcosine oxidase operon, which we have named SouR. SouR is the first bacterial regulator shown to respond to sarcosine, and it is required for growth on sarcosine. Sarcosine is found in its free form and is also an intermediate in the catabolic pathways of glycine betaine, carnitine, creatine, and glyphosate. The similarity of SouR to the regulators of carnitine and glycine betaine catabolism suggests evolutionary diversification within this regulatory family to allow response to structurally similar but physiologically distinct ligands.

The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome throughout the world. A defining feature of EHEC pathogenesis is the formation of attaching and effacing (AE) lesions on colonic epithelial cells. Most of the genes that code for AE lesion formation, including a type three secretion system (T3SS) and effectors, are carried within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). In this study, we report that a putative regulator, which is encoded in the cryptic E. coli type three secretion system 2 (ETT2) locus and herein renamed EtrB, plays an important role in EHEC pathogenesis. The etrB gene is expressed as a monocistronic transcript, and EtrB autoregulates expression. We provide evidence that EtrB directly interacts with the ler regulatory region to activate LEE expression and promote AE lesion formation. Additionally, we mapped the EtrB regulatory circuit in EHEC to determine a global role for EtrB. EtrB is regulated by the transcription factor QseA, suggesting that these proteins comprise a regulatory circuit important for EHEC colonization of the gastrointestinal tract.

Effects of acoustic streaming from moderate-intensity pulsed ultrasound for enhancing biofilm mitigation effectiveness of drug-loaded liposomes.

Because biofilms have resistance to antibiotics, their control using minimum amounts of chemicals and energy becomes a critical issue particularly for resource-constrained long-term space and deep-sea explorations. This preliminary study investigates how ultrasound promoting penetration of antibiotic-loaded liposomes into alginate-based bacterial biofilms, resulting in enhanced bacterial (Ralstonia insidiosa) killing. Nano-sized liposomes are used as a delivery vehicle for the antibiotic gentamicin. Alginate-based synthetic biofilms, which are widely acknowledged as biofilm phantoms, filled with liposome solution are formed at the bottoms of six-well Petri dishes and exposed to ultrasound (frequency = 2.25 MHz, 10% duty cycle, and spatially and temporally averaged intensity ISAPA = 4.4 W/cm(2)). Gentamicin is released from liposomes after they are lysed using detergent solution (0.05% sodium dodecyl sulfate, 1.0% Triton X-100) and incubated for 20 min. The alginate biofilm is dissolved and diluted, counting of colony-forming units shows about 80% of the bacteria are killed. It has also been shown the liposome-capture density by the alginate film increases linearly with the ultrasound intensity up to ISAPA = 6.2 W/cm(2) reaching approximately threefold that without ultrasound. Measurement by using particle-image velocimetry has demonstrated the acoustic streaming with modification by thermal convection controls the enhancement of the liposome capture rate.

The yad and yeh fimbrial loci influence gene expression and virulence in enterohemorrhagic Escherichia coli O157:H7.

Fimbriae are essential virulence factors for many bacterial pathogens. Fimbriae are extracellular structures that attach bacteria to surfaces. Thus, fimbriae mediate a critical step required for any pathogen to establish infection by anchoring a bacterium to host tissue. The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7encodes 16 fimbriae that may be important for EHEC to initiate infection and allow for productive expression of virulence traits important in later stages of infection, including a type III secretion system (T3SS) and Shiga toxin; however, the roles of most EHEC fimbriae are largely uncharacterized. Here, we provide evidence that two EHEC fimbriae, Yad and Yeh, modulate expression of diverse genes including genes encoding T3SS and Shiga toxin and that these fimbriae are required for robust colonization of the gastrointestinal tract. These findings reveal a significant and previously unappreciated role for fimbriae in bacterial pathogenesis as important determinants of virulence gene expression.IMPORTANCEFimbriae are extracellular proteinaceous structures whose defining role is to anchor bacteria to surfaces. This is a fundamental step for bacterial pathogens to establish infection in a host. Here, we show that the contributions of fimbriae to pathogenesis are more complex. Specifically, we demonstrate that fimbriae influence expression of virulence traits essential for disease progression in the intestinal pathogen enterohemorrhagic Escherichia coli. Gram-positive and Gram-negative bacteria express multiple fimbriae; therefore, these findings may have broad implications for understanding how pathogens use fimbriae, beyond adhesion, to initiate infection and coordinate gene expression, which ultimately results in disease.

Structure of a human monoclonal antibody in complex with Outer surface protein C (OspC) of the Lyme disease spirochete, Borreliella burgdorferi.

Lyme disease is a tick-borne, multisystem infection caused by the spirochete, Borreliella burgdorferi . Although antibodies have been implicated in the resolution of Lyme disease, the specific B cell epitopes targeted during human infections remain largely unknown. In this study, we characterized and defined the structural epitope of a patient-derived bactericidal monoclonal IgG ("B11") against Outer surface protein C (OspC), a homodimeric lipoprotein necessary for B. burgdorferi tick-mediated transmission and early-stage colonization of vertebrate hosts. High-resolution epitope mapping was accomplished through hydrogen deuterium exchange-mass spectrometry (HDX-MS) and X-ray crystallography. Structural analysis of B11 Fab-OspC A complexes revealed the B11 Fabs associated in a 1:1 stoichiometry with the lateral faces of OspC A homodimers such that the antibodies are essentially positioned perpendicular to the spirochete's outer surface. B11's primary contacts reside within the membrane proximal regions of α-helices 1 and 6 and adjacent loops 5 and 6 in one OspC A monomer. In addition, B11 spans the OspC A dimer interface, engaging opposing α-helix 1', α-helix 2', and loop 2-3' in the second OspC A monomer. The B11-OspC A structure is reminiscent of the recently solved mouse transmission blocking monoclonal IgG B5 in complex with OspC A , indicating a mode of engagement with OspC that is conserved across species. In conclusion, we provide the first detailed insight into the interaction between a functional human antibody and an immunodominant Lyme disease antigen long considered an important vaccine target.

Flagellar-based motility accelerates IgA-mediated agglutination of Salmonella Typhimurium at high bacterial cell densities.

Introduction: Secretory IgA (SIgA) protects the intestinal epithelium from enteric pathogens such as Salmonella enterica serovar Typhimurium (STm) through a process known as immune exclusion, where invading bacteria are aggregated via antibody cross-linking, encased in mucus, and then cleared from the intestinal tract via peristalsis. At high cell densities, the STm aggregates form a tightly packed network that is reminiscent of early bacterial biofilms. However, the underlying mechanism of how SIgA mediates this transition from a motile and invasive state to an avirulent sessile state in STm is currently unknown. Methods: In this report, we developed and validated a methodology known as the "snow globe" assay to enable real-time imaging and quantification of STm agglutination by the mouse monoclonal IgA Sal4. Results: We observed that agglutination in the snow globe assay was dose-dependent, antigen-specific, and influenced by antibody isotype. We determined that flagellar-based motility was a prerequisite for rapid onset of agglutination, even at high cell densities where cell-cell contacts are expected to be frequent. We also investigated the roles of individual cyclic-di-GMP metabolizing enzymes previously implicated in motility and biofilm formation in Sal4 IgA-mediated agglutination. Discussion: Taken together, our results demonstrate that IgA-mediated agglutination is a dynamic process influenced by bacterial motility and cell-cell collisions. We conclude that the snow globe assay is a viable platform to further decipher the molecular and genetic determinants that drive this interaction.

Cooperative interactions in the West Nile virus mutant swarm.

BACKGROUND: RNA viruses including arthropod-borne viruses (arboviruses) exist as highly genetically diverse mutant swarms within individual hosts. A more complete understanding of the phenotypic correlates of these diverse swarms is needed in order to equate RNA swarm breadth and composition to specific adaptive and evolutionary outcomes. RESULTS: Here, we determined clonal fitness landscapes of mosquito cell-adapted West Nile virus (WNV) and assessed how altering the capacity for interactions among variants affects mutant swarm dynamics and swarm fitness. Our results demonstrate that although there is significant mutational robustness in the WNV swarm, genetic diversity also corresponds to substantial phenotypic diversity in terms of relative fitness in vitro. In addition, our data demonstrate that increasing levels of co-infection can lead to widespread strain complementation, which acts to maintain high levels of phenotypic and genetic diversity and potentially slow selection for individual variants. Lastly, we show that cooperative interactions may lead to swarm fitness levels which exceed the relative fitness levels of any individual genotype. CONCLUSIONS: These studies demonstrate the profound effects variant interactions can have on arbovirus evolution and adaptation, and provide a baseline by which to study the impact of this phenomenon in natural systems.

Experimental Evolution of West Nile Virus at Higher Temperatures Facilitates Broad Adaptation and Increased Genetic Diversity.

West Nile virus (WNV, Flaviviridae, Flavivirus) is a mosquito-borne flavivirus introduced to North America in 1999. Since 1999, the Earth's average temperature has increased by 0.6 °C. Mosquitoes are ectothermic organisms, reliant on environmental heat sources. Temperature impacts vector-virus interactions which directly influence arbovirus transmission. RNA viral replication is highly error-prone and increasing temperature could further increase replication rates, mutation frequencies, and evolutionary rates. The impact of temperature on arbovirus evolutionary trajectories and fitness landscapes has yet to be sufficiently studied. To investigate how temperature impacts the rate and extent of WNV evolution in mosquito cells, WNV was experimentally passaged 12 times in Culex tarsalis cells, at 25 °C and 30 °C. Full-genome deep sequencing was used to compare genetic signatures during passage, and replicative fitness was evaluated before and after passage at each temperature. Our results suggest adaptive potential at both temperatures, with unique temperature-dependent and lineage-specific genetic signatures. Further, higher temperature passage was associated with significantly increased replicative fitness at both temperatures and increases in nonsynonymous mutations. Together, these data indicate that if similar selective pressures exist in natural systems, increases in temperature could accelerate emergence of high-fitness strains with greater phenotypic plasticity.

Recombinant Human Secretory IgA Induces Salmonella Typhimurium Agglutination and Limits Bacterial Invasion into Gut-Associated Lymphoid Tissues.

As the predominant antibody type in mucosal secretions, human colostrum, and breast milk, secretory IgA (SIgA) plays a central role in safeguarding the intestinal epithelium of newborns from invasive enteric pathogens like the Gram-negative bacterium Salmonella enterica serovar Typhimurium (STm). SIgA is a complex molecule, consisting of an assemblage of two or more IgA monomers, joining (J)-chain, and secretory component (SC), whose exact functions in neutralizing pathogens are only beginning to be elucidated. In this study, we produced and characterized a recombinant human SIgA variant of Sal4, a well-characterized monoclonal antibody (mAb) specific for the O5-antigen of STm lipopolysaccharide (LPS). We demonstrate by flow cytometry, light microscopy, and fluorescence microscopy that Sal4 SIgA promotes the formation of large, densely packed bacterial aggregates in vitro. In a mouse model, passive oral administration of Sal4 SIgA was sufficient to entrap STm within the intestinal lumen and reduce bacterial invasion into gut-associated lymphoid tissues by several orders of magnitude. Bacterial aggregates induced by Sal4 SIgA treatment in the intestinal lumen were recalcitrant to immunohistochemical staining, suggesting the bacteria were encased in a protective capsule. Indeed, a crystal violet staining assay demonstrated that STm secretes an extracellular matrix enriched in cellulose following even short exposures to Sal4 SIgA. Collectively, these results demonstrate that recombinant human SIgA recapitulates key biological activities associated with mucosal immunity and raises the prospect of oral passive immunization to combat enteric diseases.

Transcriptional profiling of Vibrio cholerae O1 following exposure to human anti- lipopolysaccharide monoclonal antibodies.

Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of the intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in gene expression that accompany antibody exposure and that may account for motility arrest and ECM production, we subjected V. cholerae O1 El Tor to RNA-seq analysis after treatment with ZAC-3 IgG, a high affinity MAb directed against the core/lipid A region of LPS. We identified > 160 genes whose expression was altered following ZAC-3 IgG treatment, although canonical outer membrane stress regulons were not among them. ompS (VCA1028), a porin associated with virulence and indirectly regulated by ToxT, and norR (VCA0182), a σ54-dependent transcription factor involved in late stages of infection, were two upregulated genes worth noting.

Inhibition of invasive salmonella by orally administered IgA and IgG monoclonal antibodies.

Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer's patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. In this report, we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to prevent infection of invasive Salmonella enterica serovar Typhimurium (STm) in mice. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer's patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer's patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer's patch tissues. Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonetheless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.