Title : Deciphering acidic stress response of brucella melitensis isolates through gene expression analysis
Abstract:
Background:
Brucellosis is a widespread zoonotic disease, endemic in the Mediterranean. Brucella can spread to humans through the consumption of raw meat, dairy products, direct contact with an infected animal, or by inhaling infected aerosols. After entering host cells, brucellae fuse with phagosomes, forming Brucella-containing vacuoles which experience transient interactions with lysosomes, leading to their acidification. The intracellular pH increases when they begin to interact with the endoplasmic reticulum, enabling the intracellular replication of the bacterium. Brucella may exploit acidic pH as stimulus for induction of genes essential for altering the biological functions of professional phagocytes and evading their killing mechanisms.
Objective:
The aim of this study was to investigate the effect of acidity levels on the expression pattern of genes from the Toxin-Antitoxin, T4 Secretion and Two Component Systems in B. melitensis isolated from humans and animals.
Methods:
Brucella isolates (27 from animals and 17 from humans) were cultivated in TSB at pH 7.2 (control), pH 4.4 and 3.4 at time intervals of 1 and 3 hours. We analyzed the expression of seven genes (virb10, cogt, fic, cbb3, otpr, rele, brnt) by RT-qPCR. Primers were designed using Primer3 Software. The integrity of the extracted [NucleoSpin RNA, Mini kit (Macherey-Nagel, Germany)] RNA was tested by agarose gel electrophoresis and the concentration was measured at a Nanodrop. The total RNA was reverse-transcribed (PrimeScript RT Reagent Kit; Takara Bio, Japan) into cDNA and tested by real time-PCR (SYBR Select Master Mix; Thermo Fisher Scientific, USA), in duplicate at a Bio-rad CFX96 Real-Time PCR cycler. The expression levels were normalized by 16S rRNA and analyzed using the 2 −ΔΔCT relative quantification method. All conditions were compared against the control (pH 7.2) by Student’s t-test. P-values were corrected for false discovery rate using the Benjamini & Hochberg method.
Results:
The genes’ expression patterns differentiated among the strains. In most of the strains, the expression levels were higher at pH 3.4 and after 3h of the effect and presented statistically significant differences. Strains exposed to pH 3.4 at both time points exhibited differentiated rates of gene expression compared to pH 4.4. Duration of exposure and different pH levels seem to influence gene expression. Some genes didn’t show any specific expression pattern. Interestingly, animal and human isolates didn’t steadily follow the same expression patterns.
Conclusion:
Although several genes were differentially expressed (over or under expressed) under specific pH and time points it was difficult to distinguish a clear gene-response pattern of the bacteria. Isolates from different hosts didn’t always share similar regulation-patterns of gene expression. The induction of certain genes may initiate the expression of others, in order to preserve Brucella’s viability.
Acknowledgments
This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call ERANETs 2021Α. Project code: T12EPA5-00064
Audience Take away:
• Present the effect of acidic stress on B.melitensis field strains
• Discuss the importance of zoonotic diseases
• Make public results that can contribute to the existing research or stimulate new research projects
