Defensin-driven viral evolution.

Enteric alpha-defensins are potent effectors of innate immunity that are abundantly expressed in the small intestine. Certain enteric bacteria and viruses are resistant to defensins and even appropriate them to enhance infection despite neutralization of closely related microbes. We therefore hypothesized that defensins impose selective pressure during fecal-oral transmission. Upon passaging a defensin-sensitive serotype of adenovirus in the presence of a human defensin, mutations in the major capsid protein hexon accumulated. In contrast, prior studies identified the vertex proteins as important determinants of defensin antiviral activity. Infection and biochemical assays suggest that a balance between increased cell binding and a downstream block in intracellular trafficking mediated by defensin interactions with all of the major capsid proteins dictates the outcome of infection. These results extensively revise our understanding of the interplay between defensins and non-enveloped viruses. Furthermore, they provide a feasible rationale for defensins shaping viral evolution, resulting in differences in infection phenotypes of closely related viruses.

Flagellin-independent effects of a Toll-like receptor 5 polymorphism in the inflammatory response to Burkholderia pseudomallei.

BACKGROUND: Toll-like receptors (TLRs) are sentinel receptors of the innate immune system. TLR4 detects bacterial lipopolysaccharide (LPS) and TLR5 detects bacterial flagellin. A common human nonsense polymorphism, TLR5:c.1174C>T, results in a non-functional TLR5 protein. Individuals carrying this variant have decreased mortality from melioidosis, infection caused by the flagellated Gram-negative bacterium Burkholderia pseudomallei. Although impaired flagellin-dependent signaling in carriers of TLR5:c.1174C>T is well established, this study tested the hypothesis that a functional effect of TLR5:c.1174C>T is flagellin-independent and involves LPS-TLR4 pathways. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood from two independent cohorts of individuals genotyped at TLR5:c.1174C>T was stimulated with wild type or aflagellated B. pseudomallei or purified bacterial motifs followed by plasma cytokine measurements. Blood from individuals carrying the TLR5:c.1174C>T variant produced less IL-6 and IL-10 in response to an aflagellated B. pseudomallei mutant and less IL-8 in response to purified B. pseudomallei LPS than blood from individuals without the variant. TLR5 expression in THP1 cells was silenced using siRNA; these cells were stimulated with LPS before cytokine levels in cell supernatants were quantified by ELISA. In these cells following LPS stimulation, silencing of TLR5 with siRNA reduced both TNF-α and IL-8 levels. These effects were not explained by differences in TLR4 mRNA expression or NF-κB or IRF activation. CONCLUSIONS/SIGNIFICANCE: The effects of the common nonsense TLR5:c.1174C>T polymorphism on the host inflammatory response to B. pseudomallei may not be restricted to flagellin-driven pathways. Moreover, TLR5 may modulate TLR4-dependent cytokine production. While these results may have broader implications for the role of TLR5 in the innate immune response in melioidosis and other conditions, further studies of the mechanisms underlying these observations are required.

Defensins Potentiate a Neutralizing Antibody Response to Enteric Viral Infection.

α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.

NLRC4 and TLR5 each contribute to host defense in respiratory melioidosis.

Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice) and type III secretion system components (in mice and humans). In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1ß production in Nlrc4(-/-) mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis.

Common TLR1 genetic variation is not associated with death from melioidosis, a common cause of sepsis in rural Thailand.

Melioidosis, infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is a common cause of sepsis in northeast Thailand. In white North Americans, common functional genetic variation in TLR1 is associated with organ failure and death from sepsis. We hypothesized that TLR1 variants would be associated with outcomes in Thais with melioidosis. We collated the global frequencies of three TLR1 variants that are common in white North American populations: rs5743551 (-7202A/G), rs4833095 (742A/G), and rs5743618 (1804G/T). We noted a reversal of the minor allele from white North American subjects to Asian populations that was particularly pronounced for rs5743618. In the Utah residents of European ancestry, the frequency of the rs5743618 T allele was 17% whereas in Vietnamese subjects the frequency was >99%. We conducted a genetic association study in 427 patients with melioidosis to determine the association of TLR1 variation with organ failure or death. We genotyped rs5743551 and rs4833095. The variants were in high linkage disequilibrium but neither variant was associated with organ failure or in-hospital death. In 300 healthy Thai individuals we further tested the association of TLR1 variation with ex vivo blood responses to Pam3CSK4, a TLR1 agonist. Neither variant was robustly associated with blood cytokine responses induced by Pam3CSK4. We identified additional common variation in TLR1 by searching public databases and the published literature and screened three additional TLR1 variants for associations with Pam3CSK4-induced responses but found none. We conclude that the genetic architecture of TLR1 variation differs substantially in southeast Asians compared to other populations and common variation in TLR1 in Thais is not associated with outcome from melioidosis or with altered blood responses to Pam3CSK4. Our findings highlight the need for additional studies of TLR1 and other innate immune genetic modulators of the inflammatory host response and determinants of sepsis in southeast Asian populations.

The role of NOD2 in murine and human melioidosis.

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytosolic pathogen recognition receptor that regulates susceptibility to a variety of infections and chronic diseases. Burkholderia pseudomallei, a facultative intracellular bacterium, causes the tropical infection melioidosis. We hypothesized that NOD2 may participate in host defense in melioidosis. We performed a series of in vitro assays and in vivo experiments and analyzed the association of human genetic variation with infection to delineate the contribution of NOD2 to the host response to B. pseudomallei. We found that transfection with NOD2 mediated NF-κB activation induced by B. pseudomallei stimulation of HEK293 cells. After low-dose inoculation with aerosolized B. pseudomallei, Nod2-deficient mice showed impaired clinical responses and permitted greater bacterial replication in the lung and dissemination to the spleen compared with wild-type mice. IL-6 and KC levels were higher in the lungs of Nod2-deficient mice. In a cohort of 1562 Thai subjects, a common genetic polymorphism in the NOD2 region, rs7194886, was associated with melioidosis, and this effect was most pronounced in women. rs7194886 was not associated with differences in cytokine production induced by whole-blood stimulation with the NOD2 ligand, muramyl dipeptide, or B. pseudomallei. To our knowledge, these findings are the first to characterize the role of NOD2 in host defense in mammalian melioidosis.

Survey of innate immune responses to Burkholderia pseudomallei in human blood identifies a central role for lipopolysaccharide.

B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.

Directed evolution of mutator adenoviruses resistant to antibody neutralization.

We incorporated a previously identified mutation that reduces the fidelity of the DNA polymerase into a human adenovirus vector. Using this mutator vector, we demonstrate rapid selection of resistance to a neutralizing anti-hexon monoclonal antibody due to a G434D mutation in hexon that precludes antibody binding. Since mutator adenoviruses can accumulate compound mutations that are unattainable using traditional random mutagenesis techniques, this approach will be valuable to the study of antivirals and host factor interactions.

Impaired TLR5 functionality is associated with survival in melioidosis.

Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR5(1174C)>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney-293 cells transfected with TLR5 and found that B. pseudomallei induced TLR5(1174C)- but not TLR5(1174T)-dependent activation of NF-κB. We tested the association of TLR5(1174C)>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR5(1174C)>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08-0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR5(1174C)>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1ß in carriers of TLR5(1174T) compared with carriers of TLR5(1174C). B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR5(1174T). We conclude that the hypofunctional genetic variant TLR5(1174C)>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis.

Murine pulmonary infection and inflammation induced by inhalation of Burkholderia pseudomallei.

Melioidosis is a tropical disease caused by ingestion, percutaneous inoculation or inhalation of the Gram-negative soil saprophyte Burkholderia pseudomallei. We developed a reproducible experimental murine model of pneumonic melioidosis induced by inhalation of aerosolized B. pseudomallei 1026b. In a series of experiments performed to bracket the lethal dose, we found that C57BL/6 mice were modestly more resistant than BALB/c mice (median lethal dose 334 CFU/lung vs 204 CFU/lung). We further characterized infection and pulmonary inflammation in C57BL/6 mice infected with a sublethal dose. We observed pulmonary replication and dissemination of bacteria to distant organs in the first days after infection, followed by bacterial containment by day 4 and no evidence of recrudescent infection for up to 2 months. We measured a robust host inflammatory response notable for a neutrophilic bronchoalveolar lavage fluid profile, elevated cytokines and chemokines in the lung and serum and scattered foci of neutrophilic infiltrates in the alveoli and in a perivascular distribution on histological analysis. We previously noted a similar pattern of inflammation in mice infected with aerosolized B. thailandensis. This report builds on the limited literature describing experimental murine pneumonic melioidosis induced by aerosol and characterizes pulmonary infection and resultant inflammation in C57BL/6 mice infected with aerosolized B. pseudomallei. This model has utility for the study of bacterial and host factors that contribute to the virulence of melioidosis.

Pathogenicity of high-dose enteral inoculation of Burkholderia pseudomallei to mice.

Melioidosis is a frequently lethal tropical infection caused by the environmental saprophyte Burkholderia pseudomallei. Although transcutaneous inoculation and inhalation are considered the primary routes of infection, suggestive clinical evidence implicates ingestion as a possible alternative route. We show that in BALB/c and C57BL/6 mice, direct gastric inoculation of high doses of B. pseudomallei causes systemic infection that may be lethal or cause chronic disseminated infection. Mice may shed bacteria in the stool for weeks after infection, and high titers of B. pseudomallei-specific IgG are detectable. This report of enteric murine melioidosis supports further consideration of this route of infection.