title: Prolonged heat stress of Lactobacillus casei GCRL163 and the impact on the cell physiology and probiotic functionality using proteomics
Creators: name: Adu, KT
Publisher: name: University of Tasmania
Year: 2019

description: Lactic acid bacteria (LAB) are exposed to several potential stressors during probiotic formulation, manufacture of fermented food and passage through the gastrointestinal tract (GIT). To understand the physiological and molecular mechanisms through which L. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (log2-fold change of 7) at 45°C. We identified a novel SecB homolog as a protein export chaperone putatively involved in post-translational translocation systems, which was down-regulated as growth temperature increased and where the modelled 3D-structure shared architectural similarities with the Escherichia coli SecB protein. Membrane lipid analyses revealed temporal changes in fatty acid composition, cyclization of oleic acid to cyclopropane and novel cyclopentenyl moieties, and reduced synthesis of vaccenic acid, at higher temperatures. An 18kDa α-crystallin domain, Hsp20 family heat shock protein was more highly up-regulated in response to heat stress compared to other molecular chaperones, suggesting this protein could be a useful biomarker of prolonged heat stress in L. casei GCRL163. Lactobacillus casei strains are non-starter lactic acid bacteria which improve cheese ripening and may interact with host intestinal cells as probiotics, for which cell surface proteins play a key role. To understand the impact of prolonged heat stress on L. casei surface proteins, three complementary methods (trypsin shaving [TS], LiCl-sucrose extraction [LS] and extracellular culture fluid [ECF] precipitation) were employed to obtain proteins and peptides from L. casei GCRL163 cells, grown in bioreactors under controlled conditions. Label-free quantitative proteomics was used to characterize the surface heat stress response. A total of 416 proteins, including 300 extra-cytoplasmic and 116 cytoplasmic proteins, were quantified as surface proteins. LS caused significantly higher cell lysis as growth temperature increased. However, most extra-cytoplasmic proteins were exclusively obtained from LS fractions, demonstrating the utility of LiCl extraction of surface proteins in a species producing no S layer. Cell wall hydrolases, adhesins, and homologues of major secreted L. rhamnosus GG proteins Msp1/p75 and Msp2/p40, were up-regulated in surface and secreted protein fractions, suggesting that cell adhesion may be impacted by heat stress. Hydrophobicity analysis indicated hydrophilicity was enhanced at sub- and supra-optimal growth temperatures. The binding-capacity of L. casei GCRL163 to human colorectal adenocarcinoma HT-29 cells also increased for heat-stressed relative to acid-stressed cells. This study demonstrates that prolonged heat stress influences cell adhesion and abundance of surface proteins, which may impact probiotic functionality. Understanding the expression of proteins associated with the cell surface at different growth phases can provide an in-depth insight into how bacteria manipulate their physiological processes to adapt under different environmental conditions. To this end, LFQ proteomics was applied to investigate L. casei GCRL163 surface proteins extracted by TS and LS at mid-exponential and stationary growth phases and also to profile the secreted ECF proteins. Our findings revealed an increased expression of some cell wall hydrolases, including BN194_23630 and SLAP domain-containing NlpC/P60 protein BN194_02820, and putative autolysins, suggesting induced cell wall hydrolysis, peptidoglycan autolysis and exopolysaccharide degradation in stationary phase. Proteins involved in sugar and peptide uptake, including oligopeptide ABC-type transporters OppA, OppA_2 and OppD_2, and phosphotransferase system proteins FruA_3 and BglP were more abundant, with GlnA and CysK induced at stationary growth phase in different protein fractions. Transcriptional regulator LytR, involved in sensory transduction and cell wall metabolism regulation, and proteins associated with cell surface structure synthesis, including exopolysaccharides such as RmlB, RmlB_2, SpsK and SpsK _2, were more abundantly expressed at stationary phase. The abundance of several proteins linked to probiotic functionality, including moonlighting proteins (Gap, Fab_2), Tig and cell wall hydrolase NlpC/P60 protein BN194_02820, suggested that host-cell interactions would be heightened at stationary growth phase. These findings establish the growth-phase dependent change in the surface sub-proteome of L. casei GCRL163 that possibly promotes cell adaptation and enhanced functionality. In addition, the regulation of prolonged heat stress response (PHSR) is important for adaptive responses that adjust cellular and molecular functions to maintain energy production and structural integrity of macromolecules and enzymes during prolonged heat stress. To investigate the induction of the regulatory proteins involved in PHSR in L. casei, a forensic analysis was performed on LFQ proteomic datasets of the CFEs, cell surface-associated (TS and LS extracts) and ECF protein fractions of L. casei GCRL163, cultured anaerobically at pH 6.5 in bioreactors to mid-exponential phase at 30 to 45°C. The analysis revealed that HrcA played a central regulatory role in PHSR while CtsR was not detected in any fraction. The RNA polymerase subunits α, β and δ were detected in CFEs and at the cell surface as highly up-regulated after culture at supra-optimal temperatures and sigma factor σ\(^A\) (σ\(^{70}\)) was moderately up-regulated in the CFEs, while sub-units β´, ω and presumptive ε were down-regulated at 45°C, suggesting modulation of RNA polymerase was involved in PHSR. YycF ortholog inhibitors, considered as attractive bactericidal agents against pathogenic bacteria, could impact the ability of L. casei GCRL163 to cope with growth at elevated temperature, as response regulator YycF_2 was induced at 45°C. Orthologs of proteins under the regulation of σ\(^B\) in Bacillus subtilis were up-regulated at 45°C, including Asp23-domain proteins Asp23_2, YqhY and BN194_17970. Several proteins associated with transcription, translation and post-translational modification were differentially modulated by thermal stress. This study shows the importance of proteomics in investigating stress response regulation, which can complement the current transcriptomic-based knowledge in lactobacilli, particularly in identifying novel condition-specific regulators. Metabolic processes, such as proteolysis, carbohydrate and amino acid metabolism, are pivotal in food biotechnology. Understanding the metabolic mechanisms underpinning the adaptation of the strain to prolonged heat stress is vital for improving bacterial functionalities. To investigate the modification of the metabolic pathways under prolonged heat stress, we further analysed the LFQ proteomic datasets of CFEs, cell surface-associated (TS and LS extracts) and ECF protein fractions of L. casei GCRL163, cultured to mid-exponential growth phase at 30°C, 40°C and 45°C and pH 6.5 in bioreactor systems. The proteomic data revealed upregulation of EIIBGal, Lev- and Man-family proteins, involved in PTS-uptake of sugars other than glucose, suggesting utilization of alternative sources of carbon. Proteins GalE_2, GalT, BGAL17, Cap4C and BN194_07390, involved in galactose and fructose metabolism were repressed. Networks of metabolic pathways were activated to channel carbon into the glycolytic pathway, including phospholipid metabolism coupled to up-regulated TpiA, pentose phosphate pathway shunt via upregulation of GntK and Gnd, tagatose metabolism by over-expressed PfkA, LacC, LacD2 and LacD2_2 and amino and nucleotide sugar metabolism by upregulation of NagA, ManD and NagB. Proteins involved in nucleotide metabolism, peptidoglycan biosynthesis and high energy-requiring fatty acid biosynthesis were repressed at 45°C, while proteins associated with RNA degradation increased. This study demonstrates that a key mechanism of PHSR in Lactobacillus spp. involves efficient management of energy generation and utilization, particularly around carbon scavenging pathways. In general, the results in this thesis provide an in-depth insight into understanding the mechanisms underlining PHSR in L. casei GCRL163 and the modulation of proteins involved in metabolic pathways and regulatory proteins that mediate PHSR in L. casei GCRL163. This thesis study has further shed more light on the impact of environmental stress conditions, including persistent heat and acid stress, on adhesion of L. casei GCRL163 to human cell lines, which can be important for probiotic strain selection.