Lansoprazolün Helicobacter pylori proteomuna etkisinin araştırılması
Investigation of the effect of Lansoprazole on the Helicobacter pylori proteome
Yazar: EDA YAZICI ÖZÇELİK
Danışman: PROF. DR. FETİYE KOLAYLI
Kocaeli Üniversitesi / Sağlık Bilimleri Enstitüsü / Tıbbi Mikrobiyoloji Ana Bilim Dalı / Mikrobiyoloji Bilim Dalı
Derece: Doktora, 2024
Konu: Mikrobiyoloji = Microbiology
Dizin: Helicobacter pylori = Helicobacter pylori

Özet:
Amaç: Helicobacter pylori (H.pylori), dünya çapında yaygın olarak görülmekle beraber Dünya Sağlık Örgütü tarafından Tip 1 kanserojen olarak tanımlanmış bir bakteridir. H.pylori'nin adezyon, kolonizasyon ve enfeksiyon oluşturmasında dış membran proteinleri oldukça önemli rol oynamaktadır. Çalışmanın amacı, antimikrobiyal etkinliği bilinen lansoprazolün H.pylori'nin  kolonizasyon ve adezyon gibi patogenezde oldukça önemli rol oynayan dış membran proteinlerine etkisinin araştırılması amaçlanmıştır.  
Yöntem: Çalışmaya H.pylori J99 standart suş ve hastalardan izole edilen iki farklı suş dahil edilmiştir. Suşların üreme eğrileri oluşturularak protein izole edileceği mid-log fazı belirlenmiştir. Lansoprazole karşı minimum inhibisyon konsantrasyonu sıvı mikrodilüsyon testi ile belirlenmiş olup bakteri kültürleri mid-log fazında iken lansoprazol uygulanmış ve  12 saat inkübe edilmiştir. Hücre peletleri biyotinilasyonu yapıldıktan sonra manyetik boncuklar ile zenginleştirilmiştir. Proteinlerin biyotinilasyonu ve zenginleştitrilmesi Western Blot analizi ile gösterilmiştir. Triptik kesimi yapılan proteinler nLC-MS/MS sistemi ile analiz edilmiştir. Elde edilen sonuçlar Proteom Discoverer 2.2 protein analiz programında incelenmiştir.
Bulgular: Çalışmada kullanılan tüm suşların mid-log fazı 42. saat olarak tespit edilmiştir. H.pylori J99, H1 suşu, H2 suşu  sub-MİK değerleri sırasıyla 8 µg/ml, 32 µg/ml, 4 µg/ml olarak belirlenmiştir. Proteom çalışması sonucunda toplam 673 adet protein tanımlanmıştır. Dış membran proteini ile ilişkili 65 protein saptanmıştır. H.pylori J99 suşunda 'Type IV secretion system apparatus protein Cag3' proteinin seviyesi artarken ' Outer membrane protein assembly factor BamA, Type IV secretion system protein, Outer membrane protein' proteinlerinin seviyesi azalmıştır. H1 suşunda 'Lipid A biosynthesis lauroyl acyltransferase (GN=EC518_11080), LPP20 lipofamily protein (GN=EC518_08370), Outer membrane protein (GN=EC518_05050), Outer membrane protein (GN=EC518_05630), Outer membrane beta-barrel protein (GN=EC518_05985), Outer membrane protein (GN=EC518_12110), TonB-dependent receptor plug domain-containing protein (GN=EC518_10990), TonB-dependent receptor (GN=EC518_12740), Ligand-gated channel (GN=EC518_05150)' proteinlerinin seviyesi artarken, 'Cag pathogenicity island protein (GN=cagT), OmpA family protein (GN=pal), Outer membrane protein (GN=EC518_13450), Type IV secretion system apparatus protein Cag3 (GN=cag3), Neuraminyllactose-binding hemagglutinin (GN=EC518_06845), Neuraminyllactose-binding hemagglutinin (GN=EC518_04795), Neuraminyllactose-binding hemagglutinin (GN=DD736_02210)' proteinlerinin seviyesi azalmıştır. Lansoprazole daha duyarlı olan H2 suşunda 'Outer membrane protein (GN=EC518_03525), Lipid A biosynthesis lauroyl acyltransferase (GN=EC518_11080), LPP20 lipofamily protein (GN=EC518_08370), Outer membrane protein (GN=EC518_13450)' proteinlerinin seviyesi artarken, 'Outer membrane protein GN=EC518_05050, Outer membrane protein (GN=EC518_05630), Outer membrane beta-barrel protein (GN=EC518_05985), Outer membrane protein (GN=EC518_12110), Outer membrane protein assembly factor BamA (GN=EC518_09480), Outer membrane beta-barrel protein (GN=EC518_12850), Outer membrane beta-barrel protein (GN=EC518_05205), Ligand-gated channel (GN=EC518_05150), TonB-dependent receptor (GN=EC518_11890), TonB-dependent receptor (GN=EC518_16180), TolC family protein (GN=EC518_04475), TolC family protein (GN=EC518_10375), Lipoprotein (GN=lpoB), Mannose-1-phosphate guanylyltransferase/mannose-6-phosphate isomerase (GN=EC518_11685)' proteinlerinin seviyesi azalmıştır. Her üç suşta da adezyondan sorumlu olan SabA ilişkili varsayımsal sekiz proteinin ekspresyonunda azalma   olduğu belirlenmiştir.
Sonuç: Lansoprazolün H.pylori proteomuna etkisi ilk kez araştırıldığı bu tez çalışmasında lansoprazol etkisi altında suşların protein profillerinin birbirinden farklılık gösterdiği belirlenmiştir. Lansoprazolün H.pylori dış membran proteinlerinin ekspresyonlarına etkili olabileceği tespit edilmiştir. Lansoprazolün özellikle H.pylori adezyonu üzerine etkinliğinin daha iyi anlaşılabilmesi için bu proteinlerin ekspresyonlarının kantitatif  PCR gibi gen ekspresyon düzeylerini belirleyen yöntemlerle değişimin gösterilmesinin uygun olacağı düşünülmektedir.

Abstract:
Object: Helicobacter pylori (H.pylori) is a bacterium that is common worldwide and has been defined as a Type 1 carcinogen by the World Health Organization. Outer membrane proteins play a very important role in the colonization, adhesion, and infection of H.pylori. The study aimed to investigate the effect of lansoprazole, which is known to have antimicrobial activity, on the outer membrane proteins of H. pylori, which play a very important role in pathogenesis such as colonization and adhesion.
Methods: H.pylori J99 standard strain and two different strains isolated from patients were included in the study. By creating growth curves of the strains, the mid-log phase from which the protein would be isolated was determined. The minimum inhibitory concentration against lansoprazole was determined by the broth microdilution test, and lansoprazole was applied when the bacterial cultures were in the mid-log phase and incubated for 12 hours. After biotinylation, cell pellets were enriched with magnetic beads. Biotinylation and enrichment of proteins were demonstrated by Western Blot analysis. Tryptic-digested proteins were analyzed by the nLC-MS/MS system. The results obtained were examined in the Proteome Discoverer 2.2 protein analysis program.
Results: The mid-log phase of all strains used in the study was determined as the 42nd hour. H. pylori J99, H1 strain, and H2 strain sub-MIC values were determined as 8 µg/ml, 32 µg/ml, and 4 µg/ml, respectively. As a result of the proteome study, a total of 673 proteins were identified. In the proteome analysis, 65 proteins related to the outer membrane of the protein were detected. In the H.pylori J99 strain, the level of 'Type IV secretion system apparatus protein Cag3' protein increased, while the level of 'Outer membrane protein assembly factor BamA, Type IV secretion system protein, Outer membrane protein' proteins decreased. In the H1 strain, 'Lipid A biosynthesis lauroyl acyltransferase (GN=EC518_11080), LPP20 lipofamily protein (GN=EC518_08370), Outer membrane protein (GN=EC518_05050), Outer membrane protein (GN=EC518_05630), Outer membrane beta-barrel protein (GN= EC518_05985), Outer membrane protein (GN=EC518_12110), TonB-dependent receptor plug domain-containing protein (GN=EC518_10990), TonB-dependent receptor (GN=EC518_12740), Ligand-gated channel (GN=EC518_05150)' proteins increase. , 'Cag pathogenicity island protein (GN=cagT), OmpA family protein (GN=pal), Outer membrane protein (GN=EC518_13450), Type IV secretion system apparatus protein Cag3 (GN=cag3), Neuraminyllactose-binding hemagglutinin (GN= EC518_06845), Neuraminyllactose-binding hemagglutinin (GN=EC518_04795), Neuraminyllactose-binding hemagglutinin (GN=DD736_02210)' proteins decreased. In the H2 strain, which is more sensitive to lansoprazole, the levels of 'Outer membrane protein (GN=EC518_03525), Lipid A biosynthesis lauroyl acyltransferase (GN=EC518_11080), LPP20 lipofamily protein (GN=EC518_08370), Outer membrane protein (GN=EC518_13450)' proteins increased, Outer membrane protein GN=EC518_05050, Outer membrane protein (GN=EC518_05630), Outer membrane beta-barrel protein (GN=EC518_05985), Outer membrane protein (GN=EC518_12110), Outer membrane protein assembly factor BamA (GN=EC518_09480), Outer membrane beta-barrel protein (GN=EC518_12850), Outer membrane beta-barrel protein (GN=EC518_05205), Ligand-gated channel (GN=EC518_05150), TonB-dependent receptor (GN=EC518_11890), TonB-dependent receptor (GN= EC518_16180), TolC family protein (GN=EC518_04475), TolC family protein (GN=EC518_10375), Lipoprotein (GN=lpoB), Mannose-1-phosphate guanylyltransferase/mannose-6-phosphate isomerase (GN=EC518_11685)' proteins decreased. It was determined that there was a decrease in the expression of eight hypothetical SabA-related proteins responsible for adhesion in all three strains.
Conclusions: The effect of lansoprazole on the H.pylori protein was examined for the first time in this thesis study. The strains used in the study had different lansoprazole sensitivities and it was observed that the effect of lansoprazole on these strains was different. In this study, where the effectiveness of lansoprazole on H.pylori was investigated, the protein profiles of the strains were found to be different from each other as a result of the proteome analysis. It has been determined that lansoprazole may affect the expression of the outer membrane proteins of H.pylori. It would be appropriate to conduct further studies on these hypothetical proteins to better understand the effectiveness of lansoprazole on H.pylori adhesion.