The BolA-like protein family is widespread among prokaryotes and eukaryotes. BolA was originally described in E. coli as a gene induced in the stationary phase and in stress conditions. The BolA overexpression makes cells spherical. It was characterized as a transcription factor modulating cellular processes such as cell permeability, biofilm production, motility, and flagella assembly. BolA is important in the switch between motile and sedentary lifestyles having connections with the signaling molecule c-di-GMP. BolA was considered a virulence factor in pathogens such as Salmonella Typhimurium and Klebsiella pneumoniae and it promotes bacterial survival when facing stresses due to host defenses.
1. The Role of BolA in E. coli Survival
BolA was discovered in
E. coli in the 1980s and its name is due to its ability to produce osmotically stable spherical cells when overexpressed. It was shown to be involved in switching the cells between elongation and septation systems
[1] during the cell division cycle, and the expression of
bolA was shown to be growth-rate regulated, being induced during the transition into stationary phase
[2][3][4]. BolA overexpression was responsible for spherical morphology in rod-shaped
E. coli cells. Later, it was shown that BolA could also be induced in the exponential phase of growth, in response to several stresses
[5][6][7]. BolA-like proteins constitute a widely conserved family of proteins widespread among prokaryotes and eukaryotes
[1][8]. Phylogenetic analyses allowed to group BolA proteins into four subfamilies: BolA1-like (present in both prokaryotes and eukaryotes), BolA2-like and BolA3-like (found in eukaryotes) and BolA4-like (present only in photosynthetic organisms)
[1][9][10][11]. A diversity of phenotypes has been linked to this protein family, although the molecular mechanisms that mediate BolA cellular effects are not yet well understood. Often, organisms encode several BolA members, performing different functions within a species and across the species. A phylogenetic tree for
bolA was constructed based on the sequences of
bolA1 genes from different species (
Figure 1). The sequences of each gene were obtained from the NCBI database
[12]. The phylogenetic reconstruction was created in MEGA software v11.0.11
[13].
Figure 1. Maximum-likelihood phylogenetic reconstructions of
bolA1 using MEGA software v11.0.11
[13]. Distance estimation was obtained by Tamura–Nei model. Numbers at the nodes represent bootstrap values (%) based on 1000 replicates. Gene sequences were aligned using MUSCLE and the trees were reconstructed using default settings. The sequences of each gene were obtained from NCBI database
[12] and the GenBank accession numbers are written next to each species.
In
E. coli, BolA is a small protein (≈12 kDa)
[14] which is induced at the stationary phase of growth and by several stresses. It has been linked to membrane permeability, motility, cell morphology and biofilm development
[1][4][5][6][7][15]. In 1988, Aldea and colleagues discovered that BolA is a
FtsZ-dependent morphogene, and its overexpression made
E. coli rod-shaped cells become spherical. This gave origin to the name of the gene:
bolA (meaning ball)
[1]. The mechanism by which BolA affects cell morphology is mediated by different factors. For instance, BolA binds to the promoter region of
mreBCD decreasing the expression of the actin-like
mreB [16]), and upregulates the genes
dacA and
dacC which codify the two main d-d-carboxypeptidases (respectively Penicillin-binding protein PBP5 and PBP6), regulating peptidoglycan biosynthesis
[1][15][17]. It was also shown that BolA controlled the transcription of
ampC (AmpC), a class C beta-lactamase, thus connecting for the first-time penicillin-binding proteins (PBPs) and beta-lactamases at the level of gene regulation
[15].
The overexpression of BolA has an influence in the outer membrane permeability of
E. coli. High levels of BolA have been shown to increase the ratio of OmpC/OmpF porins, turning the cell less permeable, and conferring protection from unfavorable environments
[5]. Overexpression of BolA could even confer protection from detergents and from the antibiotic Vancomycin.
BolA is controlled at transcriptional, post-transcriptional and post-translational levels. Transcription of
bolA can start at two different promoters. P2 is a constitutive promoter that is under the control of σ
70 and is detectable in low amounts during all stages of growth. P1 is located 80 nt downstream, is under the control of σ
S and is expressed in stationary phase or under stress conditions
[4][6]. For instance, in the face of stress conditions such as heat shock and acidic stress,
bolA1p mRNA levels are increased
[6]. Heat shock induction is almost immediate while the acidic stress is associated with a more gradual induction of
bolA mRNA. In response to carbon starvation and osmotic shock
bolA1p is highly induced and the level of its expression can largely exceed the ones reached in the stationary phase. These stresses make cells change their morphology to a rounder shape similar to those cells in which BolA is overexpressed in the stationary phase. On the other hand, oxidative stress leads to a moderate increase in mRNA
bolA1p levels inhibiting growth and viability
[6]. H-NS, a histone-like protein, was found to negatively regulate
bolA expression in vivo and to interact with both
bolA1p and
bolA2p regions in vitro
[18]. OmpR, in its phosphorylated form (phospho-OmpR), binds to the OmpR-binding region of
bolA1, repressing its transcription
[19]. Endoribonuclease RNase III acts as a post-transcriptional modulator of
bolA expression under carbon-starvation conditions
[20]. RNase III positively regulates
bolA1p mRNA levels and stabilities. RNase III is furthermore shown to be necessary for the normal expression of σ
S, ensuring normal levels of
rpoS mRNA and σ
S protein under glucose starvation. Accordingly, under this stress,
bolA transcript is increased and is more stable. This shows that
bolA transcriptional and post-transcriptional controls are consonant to achieve the global regulation of the expression of this gene
[5]. In 1997, Cao and Sarkar discovered that poly (A)-polymerase was able to directly regulate mRNA levels of both
bolA and
rpoS, and
bolA transcript could be polyadenylated at its 3′end
[20][21].
2. The Role of BolA in Virulence
BolA could be involved in different pathways directly related to bacterial virulence
[22].
Salmonella enterica serovar Typhimurium (
S. Typhimurium) is a pathogen that makes use of several virulence factors in order to overcome host defenses surviving inside host cells
[23][24]. In order to unravel the role of BolA protein in the virulence of
S. Typhimurium, the greater wax moth
Galleria mellonella, was used as the infection model.
G. mellonella has been extensively used as a model organism for a wide range of bacterial species including
S. Typhimurium. BolA proved to be a determinant factor in the virulence capacity of
S. Typhimurium and in its ability to survive and overcome host defenses. It conferred resistance to acidic and oxidative stress promoting its survival under harsh conditions
[25]. When cells were infected with
S. Typhimurium the wild-type bacteria could survive and multiply but the number of bacteria inside each cell was substantially reduced in the
S. Typhimurium
bolA deletion mutant.
To further explore the role of BolA in virulence,
S. Typhimurium metabolism was investigated. Using 1H-NMR metabolomics, the metabolic differences between strains expressing different levels of BolA in a minimal virulence-inducing medium (LPM medium) were accessed. The strain overexpressing BolA revealed increased levels of acetate, valine, alanine, NAD+, succinate, coenzyme A, glutathione, and putrescine. These metabolites are implicated in pathways related to stress resistance and virulence. This suggests that BolA has an important role in metabolic regulation and that potentiates the virulence of
S. Typhimurium
[26].
Recently, BolA has also been identified as a virulence factor in
Klebsiella pneumoniae [27] K. pneumonia bolA deletant mutants are less resistant to bile and oxidative stresses than wild-type cells. BolA is required for maintaining a proper cell morphology in the stationary phase of growth. In a
Galleria melonella infection model, the larvae infected with
bolA deletant
K. pneumoniae, survived 53% more than the larvae infected with wild-type strain. BolA promoted the adhesion of
K. pneumoniae to human cancer epithelial cells and significantly decreased the bacterial ability to colonize the liver, spleen, lung and kidney organs in a mouse model. Additionally, the formation of liver abscesses was not observed in mice infected with
bolA deletant
K. pneumoniae. BolA positively regulated siderophores production and biofilm formation as well as metabolites related to stress response and virulence (agmatine, cadaverine, guanosine, flavin adenine dinucleotide [FAD] and
d-biotin). According to the authors, the downregulation of these metabolites may be the factor leading to the loss of virulence and stress resistance of the Δ
bolA strain of
K. pneumoniae [27].