Listeria monocytogenes is a facultative pathogenic agent of listeriosis, which is a foodborne infection in the older adults, children, and immunocompromised patients. L. monocytogenes demonstrates global distribution with cases throughout Europe, North America, Oceania, Asia, South America, and Africa, highlighting its pervasive presence worldwide (Moura et al., 2021; Koopmans et al., 2023). The annual incidence of listeriosis ranges from 0.1 to 11.3 cases per million individuals, with variations caused by geographical areas and the monitoring techniques that are used (Koopmans et al., 2023). L. monocytogenes enters the host and survives a series of potentially lethal conditions in the digestive tract, including gastric acid, biliary salt, and oxidative stress, eventually causing bacteremia, meningitis, and severe maternal–fetal infections (Allerberger and Wagner, 2010). The South African listeriosis outbreak in 2017–2018, caused by ST6 L. monocytogenes, resulted in 204 fatalities and over $260 million in fatality-related costs (Olanya et al., 2019). L. monocytogenes has been found in a diverse range of sources, with a notable predominance in contaminated food products such as processed meats, soft cheeses, raw milk, and ready-to-eat foods (Ryser and Marth, 2007). This pathogen is also extensively found in natural environmental reservoirs, such as soil and polluted water systems (Ryser and Marth, 2007).

Listeria monocytogenes can be classified into 4 lineages, 13 recognized serotypes, and numerous sequence types (STs). Certain clones of L. monocytogenes have been verified and are more closely linked to clinical cases or food sources. Lineage I strains are used to form a majority of clinical isolates, while lineage II strains exhibits a more general distribution in food isolates (Maury et al., 2016). ST1, ST2, ST4, and ST6 clones are linked to clinical isolates, while ST9 and ST121 clones are common isolates in food sources (Maury et al., 2016; Muchaamba et al., 2021). Additionally, ST87 isolates have been identified as a clinical clone type in China (Wang et al., 2015; Li et al., 2018). The molecular characteristics of L. monocytogenes have been shown to be highly divergent in food surveillance, clinical cases, animals, and environment (Maury et al., 2016; Muchaamba et al., 2021). Although some genetic elements in a specific group were associated with virulence and/or stress resistance phenotypes, such as LIPI-4 in CC4, LGI-1 in CC8, SSI-2 in ST121, and pLMST6 in CC6, the underlying causes behind the observed epidemiological distribution and phenotypic variations in L. monocytogenes remain unexplained (Bergholz et al., 2018).

As a facultative bacterial pathogen, L. monocytogenes possesses remarkable regulatory networks in response to various conditions, such as abrupt environmental changes and host-induced stresses (Guariglia-Oropeza et al., 2014). The transcriptional regulation in bacteria serves as a crucial mechanism for their rapid adaptation and reproduction in an unpredictable environment. Sigma factors are initiation factors that induce the binding of RNA polymerase to promoters, thereby enabling cells to precisely regulate the timing and specificity of gene expression (Feklistov et al., 2014). As a central regulator, the sigma factor B (SigB) has been demonstrated to regulate a factor consisting of approximately 300 genes related to stress response, pathogenesis, cellular homeostasis, and metabolism in L. monocytogenes (Chaturongakul et al., 2011; Liu et al., 2017).

Analysis of sigB in 6 Listeria species comprising 4,390 isolates showed that non-synonymous mutations occurred less frequently than synonymous mutations in 164 sigB allelic types (Liao et al., 2017). The regulator sigB is a relatively stable gene in Listeria that has not significantly undergone evolution due to positive selection and homologous recombination (Liao et al., 2017). However, as the regulatory center of large functional networks, SigB has several characteristics that have not been explored yet, such as the specific types of sigB alleles and their unknown biological significance.

In this study, we conducted a large-scale genetic analysis of the sigB gene in 46,921 L. monocytogenes isolates and associated sigB with background information about its host bacteria. To evaluate the biological function of two dominant SigB factors, we further performed chromosomal mutation modification, cell culture assays, and survival phenotype analysis.

L. monocytogenes is an important foodborne pathogen that can colonize and penetrate the gastrointestinal tract, blood–brain barrier, and placental barrier. L. monocytogenes develops numerous stress response mechanisms in order to survive and persist in a variety of harsh environments, both outside and within the host. SigB, an alternative general stress sigma factor, modulates the transcriptional landscapes of stress response, pathogenesis, and homeostasis in L. monocytogenes (Garner et al., 2006; Toledo-Arana et al., 2009; van Der Veen and Abee, 2010; Gahan and Hill, 2014; Dorey et al., 2019). Transcription factors bind to the specific DNA sequences of the target gene and then control the initiation or inhibition of their transcription, regulating the gene expression in bacteria (Balleza et al., 2009). Mutations of bacterial transcription factors, such as K259 lysine acetylation of HrdB, F318A mutation of σ54, I48S in region 1.1 of E. coli σ70, and dynamic variation of MarR transcription factors, affect transcription regulation by altering their interaction with the RNAP core enzyme and its binding activity toward target promoters (Wigneshweraraj et al., 2002; Deochand and Grove, 2017; Kim et al., 2020; Pletnev et al., 2020). The novel SigB (Q225P) mutation in Staphylococcus aureus maintains virulence but enhances biofilm formation by affecting the promoter activity (Liu et al., 2018). The study of variation in transcription factors in bacteria can offer important insights into gene expression regulation and bacterial responses.

In this study, we demonstrated that the correlation between SigB_T1 and lineage I/III as well as SigB_T2 and lineage II, and codon 216 of SigB play an important role in the invasion and environmental stress adaptation of L. monocytogenes. The 216 variant site of SigB is located in domain sigma70 r4, which forms the second largest interface with RNA polymerase and serves as a key contact point for transcriptional activators that recognize the upstream sequences of the -35 promoter region (Paget, 2015).

In this study, 92.39% of isolates were classified as lineage I in L. monocytogenes isolates carrying SigB_T1, and 98.93% of the isolates were identified as lineage II among the isolates harboring SigB_T2. We found that L. monocytogenes with SigB_T1 had a higher invasive ability to human colon cancer cells than isolates with SigB_T2, suggesting that SigB_T1 is associated with the high virulence of L. monocytogenes. When L. monocytogenes reaches the host intestinal lumen, transcriptional reshaping is activated by SigB-mediated virulence genes (Toledo-Arana et al., 2009). SigB-mediated control of inlA and inlB expression is critical for L. monocytogenes invasion of host epithelial cells, as demonstrated by a significantly reduced invasion in a sigB deletion mutant strain (Kazmierczak et al., 2003; Kim et al., 2005; Garner et al., 2006; Bierne et al., 2007). InlA is essential for the pathogenesis of L. monocytogenes as it mediates the identification and invasion of epithelial cells through its specific interaction with E-cadherin, which is a fundamental step to crossing the intestinal barrier (Bierne et al., 2007; Disson et al., 2008). Among 15 transcriptional regulators, SigB is a key factor in the invasion of human epithelial colorectal adenocarcinoma cells by L. monocytogenes (Rukit et al., 2022). Therefore, SigB_T1 is associated with the prevalence of lineage I and lineage III of L. monocytogenes in patients and animals.

Researchers in the field of the molecular epidemiology of L. monocytogenes have focused on investigating the source of human infection as well as its presence in food and food processing environments, with lineage I being frequently associated with human infection and lineage II being prevalent in food and environmental sources (Maury et al., 2016; Koopmans et al., 2023). However, little attention has been paid to its prevalence in natural environments. This study revealed that L. monocytogenes isolates in natural vs. food environments have distinct molecular characteristics, with lineage I being more prevalent in natural environments and lineage II being more prevalent in food environments. Additionally, in this study, a substantial proportion of lineage III strains originated from natural environments and animal sources. These are likely due to two distinct environmental conditions: natural environments have limited nutrition, while food environments have abundant nutrition.

The sigB gene plays a crucial role in biofilm formation and has increased expression levels in both static and continuous-flow biofilms (van Der Veen and Abee, 2010; Hsu et al., 2020). Our findings suggest that SigB_T1 enhances biofilm formation in low-nutrient settings in the MWB culture medium. The effect of SigB factor types on clinical strain LM188 is significantly apparent compared to that on strain EGD-e. This might be attributed to weaker phenotypes of EGD-e, which is a strain of unclear source, specifically biofilm formation and reduced growth in defined minimal media. Therefore, SigB_T1 contributes to the higher biofilm-forming ability observed in lineage I strains under such conditions, which may partially explain its prevalence and persistence in natural environments.

The sigB gene is involved in survival from chill stress in L. monocytogenes, with varying degrees of impact influenced by different serotypes (Moorhead and Dykes, 2004). Our finding also indicates that SigB_T1 isolates exhibit a greater capacity for cold tolerance as compared to SigB_T2 isolates when cultured in a minimal medium. This observation suggests that SigB_T1 would help lineage I and lineage III isolates endure low temperatures under nutrient-poor conditions and increase their likelihood of survival and persistence in frigid natural environments. L. monocytogenes ubiquitously exist in various sources and can survive in the presence of high osmolarity, low-temperature conditions, and extreme environments (Chan and Wiedmann, 2009). Different microbial lineages have adapted to different environments and sources, and their proportions vary depending on the environment they inhabit.

In conclusion, our study demonstrated that lineage-specific SigB types influenced the survival, virulence, and adaptation of L. monocytogenes under specific conditions. Contrary to SigB_T2, SigB_T1 exhibited a more pronounced role in promoting invasiveness and virulence as well as improving biofilm formation and long-term cold survival under limited nutritional conditions. SigB_T1 could contribute to the prevalence and persistence of L. monocytogenes linage I and linage III strains in clinical settings and the natural environment, highlighting its pivotal role in the epidemiology of this pathogen. These findings offer a new insight into the molecular mechanisms underlying the phenotypic variations and epidemiological distribution of L. monocytogenes, paving the way for future research on the development of targeted interventions to prevent and control infections caused by this pathogen.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

CY: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing—review & editing. PM: Conceptualization, Formal analysis, Methodology, Software, Visualization, Writing—original draft. YW: Supervision, Project administration, Writing—review & editing. LG: Data curation, Writing—review & editing. LLiu: Methodology, Writing—original draft. JC: Methodology, Writing—original draft. LLi: Methodology, Writing—original draft. HS: Project administration, Writing—review & editing. XL: Project administration, Writing—review & editing.