Pradeep Kumar Kondadi

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

BACKGROUND: The canine Gram-negative Helicobacter bizzozeronii is one of seven species in Helicobacter heilmannii sensu lato that are detected in 0.17-2.3% of the gastric biopsies of human patients with gastric symptoms. At the present, H. bizzozeronii is the only non-pylori gastric Helicobacter sp. cultivated from human patients and is therefore a good alternative model of human gastric Helicobacter disease. We recently sequenced the genome of the H. bizzozeronii human strain CIII-1, isolated in 2008 from a 47-year old Finnish woman suffering from severe dyspeptic symptoms. In this study, we performed a detailed comparative genome analysis with H. pylori, providing new insights into non-pylori Helicobacter infections and the mechanisms of transmission between the primary animal host and humans. RESULTS: H. bizzozeronii possesses all the genes necessary for its specialised life in the stomach. However, H. bizzozeronii differs from H. pylori by having a wider metabolic flexibility in terms of its energy sources and electron transport chain. Moreover, H. bizzozeronii harbours a higher number of methyl-accepting chemotaxis proteins, allowing it to respond to a wider spectrum of environmental signals. In this study, H. bizzozeronii has been shown to have high level of genome plasticity. We were able to identify a total of 43 contingency genes, 5 insertion sequences (ISs), 22 mini-IS elements, 1 genomic island and a putative prophage. Although H. bizzozeronii lacks homologues of some of the major H. pylori virulence genes, other candidate virulence factors are present. In particular, we identified a polysaccharide lyase (HBZC1_15820) as a potential new virulence factor of H. bizzozeronii. CONCLUSIONS: The comparative genome analysis performed in this study increased the knowledge of the biology of gastric Helicobacter species. In particular, we propose the hypothesis that the high metabolic versatility and the ability to react to a range of environmental signals, factors which differentiate H. bizzozeronii as well as H. felis and H. suis from H. pylori, are the molecular basis of the of the zoonotic nature of H. heilmannii sensu lato infection in humans.

Terminal sialic acid in the lipopolysaccharides (LPSs) of mucosal pathogens is an important virulence factor. Here we report the characterization of a Helicobacter sialyltransferase involved in the biosynthesis of sialylated LPS in Helicobacter bizzozeronii, the only non-pylori gastric Helicobacter species isolated from humans thus far. Starting from the genome sequences of canine and human strains, we identified potential sialyltransferases downstream of three genes involved in the biosynthesis of N-acetylneuraminic acid. One of these candidates showed monofunctional α,2,3-sialyltransferase activity with a preference for N-acetyllactosamine as a substrate. The LPSs from different strains were shown by SDS-PAGE and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to contain sialic acid after neuraminidase treatment. The expression of this sialyltransferase and sialyl-LPS appeared to be a phase-variable characteristic common to both human and canine H. bizzozeronii strains. The sialylation site of the LPSs of two H. bizzozeronii strains was determined to be NeuAc-Hex-HexNAc, suggesting terminal 3'-sialyl-LacNAc. Moreover, serological typing revealed the possible presence of sialyl-Lewis X in two additional strains, indicating that H. bizzozeronii could also mimic the surface glycans of mammalian cells. The expression of sialyl-glycans may influence the adaptation process of H. bizzozeronii during the host jump from dogs to humans.

Genomic analysis of a metronidazole resistant H. bizzozeronii strain revealed a frame length extension of the oxygen-insensitive NAD(P)H-nitroreductase HBZC1_00960 (RdxA), associated with the disruption of the C-terminal cysteine-containing conserved region (IACLXALGK). This was the result of the extension (from C8 to C9) of a simple sequence cytosine repeat (SSCR) located in the 3' of the gene. A 3' SSCR is also present in the rdxA homolog of H. heilmannii sensu stricto, but not in H. pylori. We showed that in the majority of in vitro spontaneous H. bizzozeronii metronidazole resistant mutants, the extension of the 3' SSCR of rdxA was the only mutation observed. In addition, we observed that H. bizzozeronii ΔrdxA mutant strain showed the same MIC value of metronidazole observed in the spontaneous mutants. These data indicate that loss of function mutations in rdxA and in particular the disruption of the conserved region IACLXALGK is associated with reduced susceptibility to metronidazole in H. bizzozeronii. Slipped-strand mispairing of the SSCR located in the 3' of the H. bizzozeronii rdxA appears to be the main mechanism. We also observed that H. bizzozeronii acquires resistance to metronidazole at high mutation rate, and that serial passages in vitro without selection induced an increased level of susceptibility. In conclusion, contrary to what was previously described in H. pylori, the H. bizzozeronii rdxA appears to be a contingency gene which undergoes phase variation. The contingency nature of rdxA should be carefully considered when metronidazole is used in the treatment of H. heilmannii-associated gastritis.

To investigate the microevolution of Helicobacter bizzozeronii in the human stomach, comparative genomics of antrum-derived populations, obtained 3 months before (T(0)) and 6 months after (T(1)) an unsuccessful eradication treatment, was performed. For each time point, the DNA of bacterial mass, representing the population diversity in three biopsies, was mixed in equal amounts and sequenced using Illumina technology. Polymorphic sites (PSs) were detected by mapping the reads against an isogenic reference genome, derived from a corpus isolate obtained at T(0). The total numbers of PSs detected in the H. bizzozeronii population at T(0) and T(1) were 128 and 223, affecting 81 and 134 coding sequences, respectively. At T(0) in 91.4% of the PSs the mutation appeared at a frequency of 50% or less. On the contrary, in the majority of the PSs observed in T(1) (71.3%) the mutation had a frequency >75%. Although only a minority of mutations were fixed in the antrum-derived population at T(0), a certain level of allelic variability, compared with the corpus-derived reference genome, was present and most likely arose as consequence of the long-term colonization of the patient. The treatment probably induced a sudden decrease of population size, selecting a subpopulation, which acted as founder for the new population at T(1) characterized by a higher number of fixed mutations. These data demonstrate that genome plasticity is an important common prerequisite among gastric Helicobacter species for adaptation to the stomach environment allowing the bacterium to evolve rapidly once a selective pressure is applied.