A prfA-like transcription factor gene lmo0753 contributes to L-rhamnose

19 Listeria monocytogenes is a foodborne bacterial pathogen and the causative agent of 20 human and animal listeriosis. Among the three major genetic lineages of L. monocytogenes (i.e. 21 LI, LII, and LIII), LI and LII are predominantly associated with foodborne listeriosis outbreaks, 22 whereas LIII is rarely implicated in human infections. In a previous study, we identified a 23 Crp/Fnr family transcription factor gene lmo0753 that was highly specific to outbreak-associated 24 LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains including a 25 DNA-binding domain with the well-characterized master virulence regulator PrfA in L. 26 monocytogenes. In this study, we constructed lmo0753 deletion and complementation mutants in 27 two fully sequenced L. monocytogenes LII strains 10403S and EGDe, and compared the flagellar 28 motility, phospholipase C production, hemolysis, and intracellular growth of the mutants and 29 their respective wild types. Our results suggested that lmo0753 plays a role in hemolytic activity 30 in both EGDe and 10403S. More interestingly, we found that deletion of lmo0753 led to the loss 31 of L-rhamnose utilization in EGDe but not in 10403S. RNA-seq analysis of EGDe Δ0753 32 incubated in Phenol-red medium containing L-rhamnose as the sole carbon source revealed that 33 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome were upand down34 regulated for more than 2-fold, respectively, when compared to the wild type strain. Genes 35 related to biotin biosynthesis, general stress response and rhamnose metabolism were shown to 36 be differentially regulated. Findings from this study collectively suggested varied functional 37 roles of lmo0753 in different LII L. monocytogenes strain backgrounds associated with human 38 listeriosis outbreaks. 39 40 on Jne 5, 2017 by gest ht://aem .sm .rg/ D ow nladed fom INTRODUCTION 41 Listeria monocytogenes is a Gram-positive, non-spore forming, facultative anaerobic 42 bacterium and the causative agent of human and animal listeriosis. Listeriosis is often caused by 43 consumption of contaminated food products such as raw milk, cheese and Ready-to-Eat (RTE) 44 meat products (1, 2). Listeriosis in healthy individuals may cause self-limiting gastroenteritis; 45 whereas the disease in immuno-compromised individuals, such as the elderly and pregnant 46 women, may cause more severe complications such as meningitis and encephalitis (1, 3, 4). Due 47 to the high rate of hospitalizations (90%) and deaths (20%) (4, 5), a zero-tolerance policy has 48 been implemented for the RTE meat and poultry industry in the U. S. (6). L. monocytogenes is 49 ubiquitous in nature and is capable of adapting to and proliferating in various environmental 50 niches such as soil, rotting vegetation, and sewage (1, 3), which makes complete eradication of 51 this persistent pathogen from industrial settings particularly challenging. 52 The species of L. monocytogenes can be phylogenetically grouped into at least three 53 major genetic lineages by various genotyping techniques (7). Lineage I (LI) mainly comprises 54 serotypes 1/2b, 3b, 4b, 4d, and 4e; lineage II (LII) contains serotypes 1/2a, 1/2c, 3a, and 3c; and 55 lineage III (LIII) consists of serotypes 4a, some 4b, and 4c (7). Previously reported human 56 listeriosis outbreaks were predominantly caused by LI and LII strains but rarely by strains in the 57 LIII group (1). In our previous study, we conducted pan-genomic analysis of 26 L. 58 monocytogenes strains, including eight from LIII (8). We identified 86 disparately distributed 59 genes (DDGs) and eight non-coding small RNAs that were highly conserved in LI and LII 60 genomes but absent in LIII genomes. The majority of these DDGs were associated with cell wall 61 structure, carbohydrate metabolism, and transcriptional regulation. We speculated that some of 62 on Jne 5, 2017 by gest ht://aem .sm .rg/ D ow nladed fom these DDGs contribute to the environmental persistence and full virulence of LI and LII strains 63 during infection, which led to major foodborne listeriosis outbreaks. 64 One interesting DDG identified from our previous study was lmo0753, which encodes a 65 putative Crp/Fnr family transcription factor. Lmo0753 shares two functional domains that are 66 specific to Crp/Fnr transcription factors, similar to the well-characterized positive regulatory 67 factor A, or PrfA. These two domains include an N-terminally located cofactor binding domain 68 and a C-terminally located helix-turn-helix Crp-type DNA binding domain (9). Crp/Fnr family 69 transcription factors function as positive regulators in bacteria and are capable of responding to 70 various environmental signals, such as anoxia, temperature, and oxidative stress (9). Due to its 71 conservation in human outbreak-associated lineages, we hypothesized that lmo0753 play a role 72 in regulating environmental survival or virulence-related mechanisms in L. monocytogenes. 73