The Lipid A Structural Heterogeneity And Molecular Mechanism In Francisella Tularensis

Lipid A (also known as endotoxin) is the major component of the outer leaflet of theouter membrane of Gram-negative bacteria. Lipid A plays an essential role in maintainingmembrane integrity and permeability and is a key factor in host innate immune recognitionand pathogenesis. Lipid A can be recognized by Toll like receptor4(TLR4), followed by theproduction of different class of cytokines, like IL-8and TNF-α. The structure of lipid A playsan important role in the immune system recognition.Francisella tularensis, an intracellularGram-negative pathogen causes the disease tularemia in animals and humans. Francisellatularensis was used as bioterrism agent. The lipid A component of LPS from Francisellasubspecies is different from those of classical enteric bacteria and cannot be recognized by theTLR4. Lipid A structure modifications plays an important role for the virulence.In this study, we demonstrate the lipid A structural heterogeneity, also the mechanisms ofstructural heterogeneity was further identified by mass spectrometry, gene expression, proteinpurification, enzyme assay, mutant construction, microarray and virulence assay. Severalnovel and observations are described in this thesis, including:(1) The mass spectrometry characterization of lipid A in F. novicida demonstratedthe lipid A structural heterogeneity was regulated by temperature.To observe the alteration of lipid A structures during transmission from an environmentalsource to a mammalian host, the lipid A synthesized by an F.novicida isolate after growth atthe representative temperatures;18oC (environment),25oC (insect), and37oC (mammalian),was analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass spectrometry (MS) in the negative ion mode. Three lipid A patterns with altereddistribution of acyl chains were identified by MALDI-TOF MS, associated with growth at18oC(m/z1609),25oC(m/z1637), and37oC(m/z1665). After growth at reduced temperatures,the size of the individual dominant peaks decreases by28Da (two methylene groups-C2H4).Dominant peaks were interrogated by MSnscanning techniques at both high andnominal mass resolution using Quadrupole ion trap-time-of-flight(QIT-TOF) MS and werefound to be complex heterogeneous mixtures where structures differed mainly at based on theN-linked hydroxy fatty acids present on the diglucosamine backbone of lipid A. ESI Q-TOFLC/MS suggested Lipid X also show the stuructural hetergenity. Lipid A kinetics remodelingin response to the temperature changed was identified by MALDI-TOF MS.(2) The identified lipid A biosynthesis genes lpxD1and lpxD2were regulated bytemperature in transcription level.Bioinformatics’ analysis of Francisella tularensis subspecies genomes identified twoN-acyltransferase enzymes responsible for the addition of N-linked acyl chains to lipid A atthe2and2’position, designated LpxD1and LpxD2. These enzymes catalyze the third step oflipid A biosynthesis, is conserved among all Gram-negative bacteria and is essential forviability. LpxD1and LpxD2share only34%amino acid sequence identity and are highlyconserved with a similar gene arrangement (>98%amino acid sequence identity) among respective Francisella tularensis subspecies homologs suggesting a conserved role in lipid Abiosynthesis in this genus. Phylogenetic analyses indicate that LpxD1shows similarity toLpxD present in enteric bacterial backgrounds, though on its own deep branch, whereas theFrancisella tularensis LpxD2is related to anaerobic bacterial backgrounds. Transcriptionalanalysis using microarray and real-time PCR confirmed that the lpxD1is up regulated at37oCand lpxD2is up regulated at21oC. Heterologous expression of the individual enzymes in E.coli confirm that these acyltransferases added long chain fatty acids to the lipid A backbone(3-OH-C16or3-OH-C18). THP-1cell stimulation results demonstrate that the modified lipidA with longer fatty acid chains was less stimulatory and the ability to be recognized by theinnate immune system was slightly decreased.(3) The enzyme assay of purified enzymes suggested LpxD1and LpxD2wereregulated by temperature in enzymatic level.To determine if acyl chain length was regulated at the enzymatic level, LpxD1andLpxD2proteins and two substrates (Acyl-ACP and UDP32-Acyl-GlcN) were purified andactivities were assayed. LpxD1enzymatic activity was2.1fold higher when assayed at37oCversus21oC. In contrast, LpxD2enzymatic activity was17.5fold higher when assayed at21oC versus37oC. Interestingly, this analysis combined with the previous transcriptionalanalyses demonstrates that the individual LpxDs are regulated by a temperature shift at boththe transcriptional and enzymatic levels. Substrate specificity assay results showed that theLpxD1enzyme preferentially added a18-carbon hydroxy fatty acid (3-OH-C18), whereas theLpxD2enzyme added a16-carbon hydroxy fatty acid (3-OH-C16). Both LpxD1and LpxD2exhibited an optimal pH at7.5, and Ca2+can inhibit the enzyme activity at100mM by bindingto the acyl-ACP.(4) The mass spectrometry characterization of lipidAisolated fromΔlpxD1andΔlpxD2deletion mutants suggested the mutant lipidAwere not regulated bytemperature.Additionally, ΔlpxD1and ΔlpxD2deletion mutant strains were made by allelic exchange.Complete lipid A structural analysis after growth at the representative temperatures;18oC(environment),25oC (insect), and37oC (mammalian)showed that the ΔlpxD1mutant lipid Awas “locked” in the environmental (m/z1609), while ΔlpxD2mutant lipid A was “locked” inthe mammalian (m/z1665). MSnanalysis suggested that the dominant lipid A peak showeddecreased heterogeneity at2and2’ position. The complemented strains recovered the lipid Aregulated phenotype at the representative temperatures. The ΔlpxD2mutant achieved similargrowth rate as the wild type F. novicida in vivo (macrophage) and in vitro (medium), ΔlpxD1mutant is slower growth at the log phase, but this mutant strain can catch up by stationaryphase of growth in vitro. Interestingly, ΔlpxD1mutant decreased the growth rate in THP-1macrophage cell line. (5) TheΔlpxD1mutant increased the cell membrane permeability and wasattenuated and protective in mice.ΔlpxD1mutants have altered outer membrane permeability, correspondence to alteredsusceptibility of some class of antimicrobial peptide and antibiotic. To investigate if a primaryinfection with the F. novicidalpxD mutant strains displayed altered virulence, a murine modelof infection was used. The ΔlpxD1mutant was attenuated in mice, as all infected micesurvived infection and showed no signs of disease. In contrast, mice infected with the WT F.novicida or ΔlpxD2mutant succumbed by day3post-infection (p.i.) Upon subsequentchallenge with the lethal wild-type organism, a protective immune response was observed.Prime-boost strategy was further used to further evaluate the protective immune response.Taken together, the individual LpxD enzymes are regulated by a temperature shift at boththe transcriptional and enzymatic levels showing that dual pathways (RNA and protein) areutilized to rapidly adapt to an environmental stress for the maintenance of bacterial membranefluidity and integrity, suggesting the importance for bacteria to finely regulate the compositionof these membrane constituents. All these results show how Francisella that survive in theenvironment prior to infecting a warm-blooded mammalian host must alter their membranearchitecture, as part of the normal pathogenic mechanism. This study may represent a generalparadigm for bacterial membrane adaptation to a mammalian host. Additionally, by restrictingthe bacteria’s ability to remodel its membrane in response to temperature may represent anovel pathway for improving the membrane permeability and decreasing the endotoxinactivity in bacteria.