Dual Transcriptional Profile of Aspergillus flavus during Co-Culture with Listeria monocytogenes and Aflatoxin B1 Production: A Pathogen–Pathogen Interaction

Abstract

The objective of this study was to investigate the effect of growth temperature and co-culture of Aspergillus flavus with Listeria monocytogenes on the production of Aflatoxin B1 (AFB1) and the transcriptional profile of associated regulatory and biosynthetic genes. The transcription of virulence- and homeostasis-associated genes of L. monocytogenes was also assessed. For this purpose, mono- and co-cultures of L. monocytogenes strain LQC 15257 and A. flavus strain 18.4 were inoculated into Malt Extract broth and allowed to grow for seven days at 25 °C and 30 °C. AFB1 quantification was performed by HPLC analysis and gene expression assessment by RT-qPCR. AFB1 production was lower at 30 °C compared to 25 °C during monoculture and also lower during co-cultures at both temperatures. This was accompanied by downregulation of aflM, aflR, aflP, and aflS during monoculture and aflM and aflS during co-culture at 30 °C. On the other hand, transcription of prfA, plcA, plcB, inlA, inlB, inlJ, murE, accA, acpP, as well as fapR, was not affected. sigB gene was downregulated after co-culture with the fungus at 25 °C and hly was downregulated after monoculture at 30 °C compared to 25 °C. In this work, the molecular interactions between A. flavus and L. monocytogenes were studied for the first time, offering a novel insight into their co-occurrence. Monitoring of their toxigenic and virulence potential at the molecular level revealed a complex dynamic in natural ecosystems.

Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

“…In total, this query returned 575 results which were manually curated to exclude those used to quantify fungal load, or those that did not specifically examine gene expression in Aspergillus , but that mentioned members of the genus in the abstract and with RT-qPCR studies in organisms other than Aspergillus . This allowed us to obtain information from 90 primary studies ( Table 1 , Table S1 ) [ 16 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , <...>…”

“…Overall, 15 species of Aspergillus were examined: A. aculeatus [ 34 , 35 ]; A. carbonarius [ 32 , 36 , 37 ]; A. cristatus [ 24 ]; A. fischeri [ 38 ]; A. flavus [ 28 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]; A. fumigatus [ 16 , 21 , 22 , 23 , 27 , 31 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 ]; A. luchuensis [ 26 , 75 ]; A. nidulans [ 30 , 76 , 77 , 78 ...…”

“…The most common reference gene used in these studies was beta-tubulin, which was used 31 times in the literature [ 38 , 41 , 42 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 76 , 80 , 81 , 82 , 83 , 98 , 106 , 109 ], followed by actin (30 times) [ 16 , 21 , 22 , 23 , 24 , 25 , 26 , 30 , 34 , 42 , 43 , 44 , 45 , 60 , 61 , 75 , 76 , 77 , 78 , 79 , 89 , 90 , 91 , 96 , 97 , 103 , 104 …”

“…We also provide an analysis of the consistency or inconsistencies of the studies regarding the validation of reference genes. Validation of specific reference genes for use under the specific experimental conditions tested was provided in only 17 of the 90 studies ( Table 1 , Table S1 ) [ 16 , 27 , 30 , 32 , 35 , 38 , 41 , 46 , 52 , 54 , 55 , 77 , 86 , 94 , 103 , 105 , 107 ]. Table 1 is organised alphabetically first by species and second by reference gene, followed by the date of publication, and includes the species examined, reference gene used, whether the reference gene was validated and the experimental conditions for each of the 90 papers returned by our PubMed search.…”

“…In this review, beta-tubulin was used as a reference gene 31 times in studies examining gene expressions in A. nidulans (5) [ 76 , 80 , 81 , 82 , 83 ], A. fumigatus (9) [ 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 ], A. flavus (13) [ 41 , 42 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ], A. parasiticus (4) [ 48 , 50 , 55 , 106 ], A. oryzae (2) [ 83 , 98 ], A. terreus (2) [ 67 , 68 ], A. westerdijkiae (1) [ 109 ] and A. fischeri (1) [ 38 ] ( Table 1 , Table S1 ). Of these 31 studies, nine provided a rationale for choosing beta-tubulin as a reference gene [ 38 , 41 , 46 , 47 , 52 , 55 , 62 , 98 , 109 ], though only six provided proper validated justification [ 38 , 41 ...…”