Modulation of the tick gut milieu by a secreted tick protein favors Borrelia burgdorferi colonization.

The Lyme disease agent, Borrelia burgdorferi, colonizes the gut of the tick Ixodes scapularis, which transmits the pathogen to vertebrate hosts including humans. Here we show that B. burgdorferi colonization increases the expression of several tick gut genes including pixr, encoding a secreted gut protein with a Reeler domain. RNA interference-mediated silencing of pixr, or immunity against PIXR in mice, impairs the ability of B. burgdorferi to colonize the tick gut. PIXR inhibits bacterial biofilm formation in vitro and in vivo. Abrogation of PIXR function in vivo results in alterations in the gut microbiome, metabolome and immune responses. These alterations influence the spirochete entering the tick gut in multiple ways. PIXR abrogation also impairs larval molting, indicative of its role in tick biology. This study highlights the role of the tick gut in actively managing its microbiome, and how this impacts B. burgdorferi colonization of its arthropod vector. Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the tick Ixodes scapularis. Here, the authors show that a tick secreted protein (PIXR) modulates the tick gut microbiota and facilitates B. burgdorferi colonization.

A Tick Antivirulence Protein Potentiates Antibiotics against Staphylococcus aureus.

New strategies are needed to combat antibiotic resistance, especially against pathogens such as methicillin-resistant Staphylococcus aureus A tick antifreeze glycoprotein, IAFGP, possesses potent antibiofilm properties against a variety of clinical pathogens, including S. aureus Synergy between IAFGP, or a peptide (P1) representative of a repeat region of the protein, with different antibiotics was assessed in vitro Antibiotics that synergized with either IAFPG or P1 were further evaluated in vivo using vertebrate and invertebrate infection models. IAFGP readily enhanced the efficacy of antibiotics against S. aureus Synergy with daptomycin, an antibiotic used to treat methicillin-resistant S. aureus, was observed in vitro and in vivo using iafgp-transgenic mice and flies. Furthermore, synergy with ciprofloxacin or gentamicin, antibiotics not generally used to treat S. aureus, was also perceived. The combined effect of the antibiotic and IAFGP was associated with improved permeation of the antibiotic into the cell. Our results highlight that synergy of IAFGP with antibiotics traditionally used to treat this pathogen, and enhancement of the potency of antibiotics not commonly used against this microbe, can provide novel alternative therapeutic strategies to combat bacterial infections.

Anti-Biofilm Activity of a Self-Aggregating Peptide against Streptococcus mutans.

Streptococcus mutans is the primary agent of dental cavities, in large part due to its ability to adhere to teeth and create a molecular scaffold of glucan polysaccharides on the tooth surface. Disrupting the architecture of S. mutans biofilms could help undermine the establishment of biofilm communities that cause cavities and tooth decay. Here we present a synthetic peptide P1, derived from a tick antifreeze protein, which significantly reduces S. mutans biofilm formation. Incubating cells with this peptide decreased biofilm biomass by approximately 75% in both a crystal violet microplate assay and an in vitro tooth model using saliva-coated hydroxyapatite discs. Bacteria treated with peptide P1 formed irregular biofilms with disconnected aggregates of cells and exopolymeric matrix that readily detached from surfaces. Peptide P1 can bind directly to S. mutans cells but does not possess bactericidal activity. Anti-biofilm activity was correlated with peptide aggregation and ß-sheet formation in solution, and alternative synthetic peptides of different lengths or charge distribution did not inhibit biofilms. This anti-biofilm peptide interferes with S. mutans biofilm formation and architecture, and may have future applications in preventing bacterial buildup on teeth.

Pathogen-mediated manipulation of arthropod microbiota to promote infection.

Arthropods transmit diverse infectious agents; however, the ways microbes influence their vector to enhance colonization are poorly understood. Ixodes scapularis ticks harbor numerous human pathogens, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We now demonstrate that A. phagocytophilum modifies the I. scapularis microbiota to more efficiently infect the tick. A. phagocytophilum induces ticks to express Ixodes scapularis antifreeze glycoprotein (iafgp), which encodes a protein with several properties, including the ability to alter bacterial biofilm formation. IAFGP thereby perturbs the tick gut microbiota, which influences the integrity of the peritrophic matrix and gut barrier-critical obstacles for Anaplasma colonization. Mechanistically, IAFGP binds the terminal d-alanine residue of the pentapeptide chain of bacterial peptidoglycan, resulting in altered permeability and the capacity of bacteria to form biofilms. These data elucidate the molecular mechanisms by which a human pathogen appropriates an arthropod antibacterial protein to alter the gut microbiota and more effectively colonize the vector.

Antivirulence properties of an antifreeze protein.

As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

Anopheles gambiae circumsporozoite protein-binding protein facilitates plasmodium infection of mosquito salivary glands.

Malaria, a mosquito-borne disease caused by Plasmodium species, causes substantial morbidity and mortality throughout the world. Plasmodium sporozoites mature in oocysts formed in the mosquito gut wall and then invade the salivary glands, where they remain until transmitted to the vertebrate host during a mosquito bite. The Plasmodium circumsporozoite protein (CSP) binds to salivary glands and plays a role in the invasion of this organ by sporozoites. We identified an Anopheles salivary gland protein, named CSP-binding protein (CSPBP), that interacts with CSP. Downregulation of CSPBP in mosquito salivary glands inhibited invasion by Plasmodium organisms. In vivo bioassays showed that mosquitoes that were fed blood with CSPBP antibody displayed a 25% and 90% reduction in the parasite load in infected salivary glands 14 and 18 days after the blood meal, respectively. These results suggest that CSPBP is important for the infection of the mosquito salivary gland by Plasmodium organisms and that blocking CSPBP can interfere with the Plasmodium life cycle.

Chelating agents exert distinct effects on biofilm formation in Staphylococcus aureus depending on strain background: role for clumping factor B.

Staphylococcus aureus is a leading cause of catheter infections, and biofilm formation plays a key role in the pathogenesis. Metal ion chelators inhibit bacterial biofilm formation and viability, making them attractive candidates as components in catheter lock solutions. The goal of this study was to characterize further the effect of chelators on biofilm formation. The effect of the calcium chelators ethylene glycol tetraacetic acid (EGTA) and trisodium citrate (TSC) on biofilm formation by 30 S. aureus strains was tested. The response to subinhibitory doses of EGTA and TSC varied dramatically depending on strain variation. In some strains, the chelators prevented biofilm formation, in others they had no effect, and they actually enhanced biofilm formation in others. The molecular basis for this phenotypic variability was investigated using two related strains: Newman, in which biofilm formation was inhibited by chelators, and 10833, which formed strong biofilms in the presence of chelators. It was found that deletion of the gene encoding the surface adhesin clumping factor B (clfB) completely eliminated chelator-induced biofilm formation in strain 10833. The role of ClfB in biofilm formation activity in chelators was confirmed in additional strains. It was concluded that biofilm-forming ability varies strikingly depending on strain background, and that ClfB is involved in biofilm formation in the presence EGTA and citrate. These results suggest that subinhibitory doses of chelating agents in catheter lock solutions may actually augment biofilm formation in certain strains of S. aureus, and emphasize the importance of using these agents appropriately so that inhibitory doses are achieved consistently.

Staphylococcus aureus clumping factor B mediates biofilm formation in the absence of calcium.

Staphylococcus aureus is the leading cause of nosocomial infections and a major cause of community-acquired infections. Biofilm formation is a key virulence determinant in certain types of S. aureus infection, especially those involving inserted medical devices. We found in a previous study that the calcium chelators sodium citrate and EGTA inhibit biofilm formation in certain strains of S. aureus but actually augment biofilm formation in other strains. Even two closely related strains, Newman and 10833, exhibited strikingly different biofilm phenotypes in the presence of calcium chelators, in that biofilm formation was inhibited in Newman but augmented in 10833. We also found that the surface protein clumping factor B (ClfB) plays a role in this phenomenon. In this study, we confirm that ClfB is required for biofilm formation under calcium-depleted conditions. We investigated the post-translational regulation of ClfB-mediated biofilm formation and found evidence that both calcium and the protease aureolysin disrupt established ClfB-dependent biofilms. Finally, we investigated the genetic basis for the biofilm-negative phenotype in strain Newman versus the biofilm-positive phenotype in strain 10833 under calcium-depleted conditions and found that strain 10833 contains a deletion that results in a stop codon within the aureolysin gene (aur). When 10833 expressed Newman aur, surface-associated ClfB and the ability to form a biofilm in chelating conditions was lost. Thus, the positive effect of chelating agents on biofilm formation in certain strains can be explained by increased ClfB activity in the absence of calcium and the discrepancy in the response of strains 10833 and Newman can be explained by point mutations in aur. This study reveals a previously unknown, to our knowledge, role for ClfB in biofilm formation and underscores the potential for striking phenotypic variability resulting from minor differences in strain background.

A low molecular weight component of serum inhibits biofilm formation in Staphylococcus aureus.

Staphylococcus aureus has a variety of genes that can influence the process of biofilm formation. The ability to establish a biofilm is an important virulence factor for this pathogen, and yet, the regulation of this process in vivo is not well understood. S. aureus can form biofilms on intravenous catheters and this process plays a key role in the pathogenesis of catheter infections. In order to investigate whether or not serum is conducive to the process of biofilm formation, we grew S. aureus in serum and analyzed biofilm thickness and expression of biofilm-related genes. Whereas serum supported planktonic bacterial growth, it was a potent inhibitor of biofilm formation. The inhibitory serum component had a molecular weight less than 3000 kDa. The component was protease-resistant and heat stable. The serum component induced a significant increase in the transcription of the intercellular adhesin gene icaA and the fibronectin binding protein gene fnbA. Transcription of other biofilm-related genes was affected in a strain-dependent manner. These results reveal that serum inhibits biofilm formation despite the fact that biofilms form on intravenous catheters. This may suggest that in vivo, biofilm formation is "selected for" by the force of blood flow and/or immune pressure rather than "induced" by serum.