Robert K. Ernst

LPS, TLR4 and infectious disease diversity

Innate immune receptors recognize microorganism-specific motifs. One such receptor–ligand complex is formed between the mammalian Toll-like receptor 4 (TLR4)–MD2–CD14 complex and bacterial lipopolysaccharide (LPS). Recent research indicates that there is significant phylogenetic and individual diversity in TLR4-mediated responses. In addition, the diversity of LPS structures and the differential recognition of these structures by TLR4 have been associated with several bacterial diseases. This review will examine the hypothesis that the variability of bacterial ligands such as LPS and their innate immune receptors is an important factor in determining the outcome of infectious disease.

Human Toll-like receptor 4 recognizes host-specific LPS modifications

Lipopolysaccharide (LPS) is the principal proinflammatory component of the Gram-negative bacterial envelope and is recognized by the Toll-like receptor 4 (TLR4)–MD-2 receptor complex. Bacteria can alter the acylation state of their LPS in response to environmental changes. One opportunistic bacterium, Pseudomonas aeruginosa, synthesizes more highly acylated (hexa-acylated) LPS structures during adaptation to the cystic fibrosis airway. Here we show that human, but not murine, TLR4–MD-2 recognizes this adaptation and transmits robust proinflammatory signals in response to hexa-acylated but not penta-acylated LPS from P. aeruginosa. Whereas responses to lipidIVA and taxol are dependent on murine MD-2, discrimination of P. aeruginosa LPS structures is mediated by an 82-amino-acid region of human TLR4 that is hypervariable across species. Thus, in contrast to mice, humans use TLR4 to recognize a molecular signature of bacterial-host adaptation to modulate the innate immune response.

Cytoplasmic LPS Activates Caspase-11: Implications in TLR4-Independent Endotoxic Shock

Move Over, TLR4 The innate immune system senses bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) (see the Perspective by Kagan). However, Kayagaki et al. (p 1246, published online 25 July) and Hagar et al. (p. 1250) report that the hexa-acyl lipid A component of LPS from Gramnegative bacteria is able to access the cytoplasm and activate caspase-11 to signal immune responses independently of TLR4. Mice that lack caspase-11 are resistant to LPS-induced lethality, even in the presence of TLR4. Cytoplasmic lipopolysaccharide from Gram-negative bacteria can activate the innate immune system directly. [Also see Perspective by Kagan] Inflammatory caspases, such as caspase-1 and -11, mediate innate immune detection of pathogens. Caspase-11 induces pyroptosis, a form of programmed cell death, and specifically defends against bacterial pathogens that invade the cytosol. During endotoxemia, however, excessive caspase-11 activation causes shock. We report that contamination of the cytoplasm by lipopolysaccharide (LPS) is the signal that triggers caspase-11 activation in mice. Specifically, caspase-11 responds to penta- and hexa-acylated lipid A, whereas tetra-acylated lipid A is not detected, providing a mechanism of evasion for cytosol-invasive Francisella. Priming the caspase-11 pathway in vivo resulted in extreme sensitivity to subsequent LPS challenge in both wild-type and Tlr4-deficient mice, whereas Casp11-deficient mice were relatively resistant. Together, our data reveal a new pathway for detecting cytoplasmic LPS.

Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium

ABSTRACT The two-component regulatory system PmrA-PmrB confers resistance ofSalmonella spp. to cationic antimicrobial peptides (AP) such as polymyxin (PM), bactericidal/permeability-increasing protein, and azurocidin. This resistance occurs by transcriptional activation of two loci termed pmrE and pmrHFIJKLM. BothpmrE and pmrHFIJKLM produce products required for the biosynthesis of lipid A with 4-aminoarabinose (Ara4N). Ara4N addition creates a more positively charged lipopolysaccharide (LPS) and thus reduces cationic AP binding. Experiments were conducted to further analyze the regulation of the pmrHFIJKLM operon and the role of this operon and the surrounding genomic region in LPS modification and antimicrobial peptide resistance. ThepmrHFIJKLM genes are cotranscribed and over 3,000-fold regulated by PmrA-PmrB. The pmrHFIJKLM promoter bound PmrA, as determined by gel shift analysis, as did a 40-bp region of the PmrA-PmrB-regulated pmrCAB promoter. Construction of nonpolar mutations in the pmrHFIJKLM genes showed that all except pmrM were necessary for the Ara4N addition to lipid A and PM resistance. The flanking genes of the operon (pmrG and pmrD) were not necessary for PM resistance, but pmrD was shown to be regulated by the PhoP-PhoQ regulatory system. BALB/c mice inoculated withpmrA and pmrHFIJKLM mutant strains demonstrated virulence attenuation when the strains were administered orally but not when they were administered intraperitoneally, indicating that Ara4N addition may be important for resistance to host innate defenses within intestinal tissues.

PmrAB, a Two-Component Regulatory System of Pseudomonas aeruginosa That Modulates Resistance to Cationic Antimicrobial Peptides and Addition of Aminoarabinose to Lipid A

Spontaneous polymyxin-resistant mutants of Pseudomonas aeruginosa were isolated. The mutations responsible for this phenotype were mapped to a two-component signal transduction system similar to PmrAB of Salmonella enterica serovar Typhimurium. Lipid A of these mutants contained aminoarabinose, an inducible modification that is associated with polymyxin resistance. Thus, P. aeruginosa possesses a mechanism that induces resistance to cationic antimicrobial peptides in response to environmental conditions.

Pseudomonas aeruginosa activates caspase 1 through Ipaf.

The innate immune system encodes cytosolic Nod-like receptors (NLRs), several of which activate caspase 1 processing and IL-1β and IL-18 secretion. Macrophages respond to Salmonella typhimurium infection by activating caspase 1 through the NLR Ipaf. This activation is mediated by cytosolic flagellin through the activity of the virulence-associated type III secretion system (T3SS). We demonstrate here that Pseudomonas aeruginosa activates caspase 1 and induces IL-1β secretion in infected macrophages. While live, virulent P. aeruginosa activate IL-1β secretion through caspase 1 and Ipaf, strains that have mutations in the T3SS or in flagellin did not. Ipaf-dependent caspase 1 activation could be recapitulated by delivering P. aeruginosa flagellin to the macrophage cytosol. We examined the role of Naip5 in P. aeruginosa-induced caspase 1 activation by using A/J (Naip5-deficient) compared with C57BL/6 and BALB/c (Naip5-sufficient) macrophages and observed that A/J macrophages secrete IL-1β in response to P. aeruginosa, S. typhimurium, and Listeria monocytogenes infection, as well as in response to cytosolic flagellin, but at slightly reduced levels. Thus, Ipaf-dependent detection of cytosolic flagellin is a conserved mechanism by which macrophages detect the presence of pathogens that use T3SS.

Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients

The opportunistic pathogen Pseudomonas aeruginosa undergoes genetic change during chronic airway infection of cystic fibrosis (CF) patients. One common change is a mutation inactivating lasR, which encodes a transcriptional regulator that responds to a homoserine lactone signal to activate expression of acute virulence factors. Colonies of lasR mutants visibly accumulated the iridescent intercellular signal 4‐hydroxy‐2‐heptylquinoline. Using this colony phenotype, we identified P. aeruginosa lasR mutants that emerged in the airway of a CF patient early during chronic infection, and during growth in the laboratory on a rich medium. The lasR loss‐of‐function mutations in these strains conferred a growth advantage with particular carbon and nitrogen sources, including amino acids, in part due to increased expression of the catabolic pathway regulator CbrB. This growth phenotype could contribute to selection of lasR mutants both on rich medium and within the CF airway, supporting a key role for bacterial metabolic adaptation during chronic infection. Inactivation of lasR also resulted in increased β‐lactamase activity that increased tolerance to ceftazidime, a widely used β‐lactam antibiotic. Loss of LasR function may represent a marker of an early stage in chronic infection of the CF airway with clinical implications for antibiotic resistance and disease progression.

Salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity.

Resistance to innate immunity is essential for salmonellae pathogenesis. The salmonellae PhoP/PhoQ regulators sense host environments to promote remodeling of the bacterial envelope. This remodeling includes enzymes that modify lipopolysaccharide (LPS). Modified LPS promotes bacterial survival by increasing resistance to cationic antimicrobial peptides and by altered host recognition of LPS.

Variation in Lipid A Structure in the Pathogenic Yersiniae

Important pathogens in the genus Yersinia include the plague bacillus Yersinia pestis and two enteropathogenic species, Yersinia pseudotuberculosis and Yersinia enterocolitica. A shift in growth temperature induced changes in the number and type of acyl groups on the lipid A of all three species. After growth at 37°C, Y. pestis lipopolysaccharide (LPS) contained the tetra‐acylated lipid IVA and smaller amounts of lipid IVA modified with C10 or C12 acyl groups, Y. pseudotuberculosis contained the same forms as part of a more heterogeneous population in which lipid IVA modified with C16:0 predominated, and Y. enterocolitica produced a unique tetra‐acylated lipid A. When grown at 21°C, however, the three yersiniae synthesized LPS containing predominantly hexa‐acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor‐α, whereas the lipid A synthesized by the three species at 37°C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperature‐dependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia, and that species‐specific lipid A forms may be important for life cycle and pathogenicity differences.

The constitutive transport element (CTE) of Mason–Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway

The constitutive transport elements (CTEs) of type D retroviruses are cis‐acting elements that promote nuclear export of incompletely spliced mRNAs. Unlike the Rev response element (RRE) of human immunodeficiency virus type 1 (HIV‐1), CTEs depend entirely on factors encoded by the host cell genome. We show that an RNA comprised almost entirely of the CTE of Mason–Pfizer monkey virus (CTE RNA) is exported efficiently from Xenopus oocyte nuclei. The CTE RNA and an RNA containing the RRE of HIV‐1 (plus Rev) have little effect on export of one another, demonstrating differences in host cell requirements of these two viral mRNA export pathways. Surprisingly, even very low amounts of CTE RNA block export of normal mRNAs, apparently through the sequestration of cellular mRNA export factors. Export of a CTE‐containing lariat occurs when wild‐type CTE, but not a mutant form, is inserted into the pre‐mRNA. The CTE has two symmetric structures, either of which supports export and the titration of mRNA export factors, but both of which are required for maximal inhibition of mRNA export. Two host proteins bind specifically to the CTE but not to non‐functional variants, making these proteins candidates for the sequestered mRNA export factors.