Corrigendum: Phenotypic and transcriptional characterization of F. tularensis LVS during transition into a viable but non-culturable state.

[This corrects the article DOI: 10.3389/fmicb.2024.1347488.].

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Phenotypic and transcriptional characterization of F. tularensis LVS during transition into a viable but non-culturable state.

Francisella tularensis is a gram-negative, intracellular pathogen which can cause serious, potentially fatal, illness in humans. Species of F. tularensis are found across the Northern Hemisphere and can infect a broad range of host species, including humans. Factors affecting the persistence of F. tularensis in the environment and its epidemiology are not well understood, however, the ability of F. tularensis to enter a viable but non-culturable state (VBNC) may be important. A broad range of bacteria, including many pathogens, have been observed to enter the VBNC state in response to stressful environmental conditions, such as nutrient limitation, osmotic or oxidative stress or low temperature. To investigate the transition into the VBNC state for F. tularensis, we analyzed the attenuated live vaccine strain, F. tularensis LVS grown under standard laboratory conditions. We found that F. tularensis LVS rapidly and spontaneously enters a VBNC state in broth culture at 37°C and that this transition coincides with morphological differentiation of the cells. The VBNC bacteria retained an ability to interact with both murine macrophages and human erythrocytes in in vitro assays and were insensitive to treatment with gentamicin. Finally, we present the first transcriptomic analysis of VBNC F. tularensis, which revealed clear differences in gene expression, and we identify sets of differentially regulated genes which are specific to the VBNC state. Identification of these VBNC specific genes will pave the way for future research aimed at dissecting the molecular mechanisms driving entry into the VBNC state.

PdpC, a secreted effector protein of the type six secretion system, is required for erythrocyte invasion by Francisella tularensis LVS.

Francisella tularensis is a gram negative, intracellular pathogen that is the causative agent of the potentially fatal disease, tularemia. During infection, F. tularensis is engulfed by and replicates within host macrophages. Additionally, this bacterium has also been shown to invade human erythrocytes and, in both cases, the Type Six Secretion System (T6SS) is required for these host-pathogen interaction. One T6SS effector protein, PdpC, is important for macrophage infection, playing a role in phagolysosomal escape and intracellular replication. To determine if PdpC also plays a role in erythrocyte invasion, we constructed a pdpC-null mutant in the live vaccine strain, F. tularensis LVS. We show that PdpC is required for invasion of human and sheep erythrocytes during in vitro assays and that reintroduction of a copy of pdpC, in trans, rescues this phenotype. The interaction with human erythrocytes was further characterized using double-immunofluorescence microscopy to show that PdpC is required for attachment of F. tularensis LVS to erythrocytes as well as invasion. To learn more about the role of PdpC in erythrocyte invasion we generated a strain of F. tularensis LVS expressing pdpC-emgfp. PdpC-EmGFP localizes as discrete foci in a subset of F. tularensis LVS cells grown in broth culture and accumulates in erythrocytes during invasion assays. Our results are the first example of a secreted effector protein of the T6SS shown to be involved in erythrocyte invasion and indicate that PdpC is secreted into erythrocytes during invasion.

Protein phosphatase SppA regulates apical growth and dephosphorylates cell polarity determinant DivIVA in Streptomyces coelicolor.

Members of the Actinobacteria, including mycobacteria and streptomycetes, exhibit a distinctive mode of polar growth, with cell wall synthesis occurring in zones at cell poles and directed by the essential cell polarity determinant DivIVA. Streptomyces coelicolor modulates polar growth via the Ser/Thr protein kinase AfsK, which phosphorylates DivIVA. Here, we show that the phosphoprotein phosphatase SppA has strong effects on polar growth and cell shape and that it reverses the AfsK-mediated phosphorylation of DivIVA. SppA affects hyphal branching and the rate of tip extension. The sppA mutant hyphae also exhibit a high frequency of spontaneous growth arrests, indicating problems with maintenance of tip extension. The phenotypic effects are partially suppressed in an afsK sppA double mutant, indicating that AfsK and SppA to some extent share target proteins. Strains with a nonphosphorylatable mutant DivIVA confirm that the effect of afsK on hyphal branching during normal growth is mediated by DivIVA phosphorylation. However, the phenotypic effects of sppA deletion are independent of DivIVA phosphorylation and must be mediated via other substrates. This study adds a PPP-family protein phosphatase to the proteins involved in the control of polar growth and cell shape determination in S. coelicolor.

The utilization of Blaptica dubia cockroaches as an in vivo model to test antibiotic efficacy.

Insects are now well recognized as biologically relevant alternative hosts for dozens of mammalian pathogens and they are routinely used in microbial pathogenesis studies. Unfortunately, these models have yet to be incorporated into the drug development pipeline. The purpose of this work was to begin to evaluate the utility of orange spotted (Blaptica dubia) cockroaches in early antibiotic characterization. To determine whether these model hosts could exhibit mortality when infected with bacteria that are pathogenic to humans, we subjected B. dubia roaches to a range of infectious doses of Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii to identify the medial lethal dose. These results showed that lethal disease did not develop following infection of high doses of S. aureus, and A. baumannii. However, cockroaches infected with E. coli and K. pneumoniae succumbed to infection (LD50s of 5.82 × 106 and 2.58 × 106 respectively) suggesting that this model may have limitations based on pathogen specificity. However, because these cockroaches were susceptible to infection from E. coli and K. pneumoniae, we used these bacterial strains for subsequent antibiotic characterization studies. These studies suggested that ß-lactam antibiotic persistence and dose was associated with reduction of hemolymph bacterial burden. Moreover, our data indicated that the reduction of bacterial CFU was directly due to the drug activity. Altogether, this work suggests that the orange-spotted cockroach infection model provides an alternative in vivo setting from which antibiotic efficacy can be evaluated.

Influence of core divisome proteins on cell division in Streptomyces venezuelae ATCC 10712.

The sporulating, filamentous soil bacterium Streptomyces venezuelae ATCC 10712 differentiates under submerged and surface growth conditions. In order to lay a solid foundation for the study of development-associated division for this organism, a congenic set of mutants was isolated, individually deleted for a gene encoding either a cytoplasmic (i.e. ftsZ) or core inner membrane (i.e. divIC, ftsL, ftsI, ftsQ, ftsW) component of the divisome. While ftsZ mutants are completely blocked for division, single mutants in the other core divisome genes resulted in partial, yet similar, blocks in sporulation septum formation. Double and triple mutants for core divisome membrane components displayed phenotypes that were similar to those of the single mutants, demonstrating that the phenotypes were not synergistic. Division in this organism is still partially functional without multiple core divisome proteins, suggesting that perhaps other unknown lineage-specific proteins perform redundant functions. In addition, by isolating an ftsZ2p mutant with an altered -10 region, the conserved developmentally controlled promoter was also shown to be required for sporulation-associated division. Finally, microscopic observation of FtsZ-YFP dynamics in the different mutant backgrounds led to the conclusion that the initial assembly of regular Z rings does not per se require the tested divisome membrane proteins, but the stability of Z rings is dependent on the divisome membrane components tested. The observation is consistent with the interpretation that Z ring instability likely results from and further contributes to the observed defects in sporulation septation in mutants lacking core divisome proteins.

OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability in Francisella tularensis LVS.

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions, including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion; however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner. We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis A fluorescently tagged OpiA (OpiA-emerald-green fluorescent protein [EmGFP]) accumulated at the cell poles of F. tularensis, which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization, and this fusion protein was detected in erythrocytes and THP-1 cells in vitro, further supporting that OpiA is secreted. Similar to previous reports with F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the proinflammatory cytokine tumor necrosis factor alpha compared to these host cells infected with wild-type bacteria. We conclude that, in addition to its role in host-pathogen interactions, our results reveal that the function of opiA is central to the biology of F. tularensis bacteria.IMPORTANCEF. tularensis is a pathogenic intracellular pathogen that is of importance for public health and strategic defense. This study characterizes the opiA gene of F. tularensis LVS, an attenuated strain that has been used as a live vaccine but that also shares significant genetic similarity to related Francisella strains that cause human disease. The data presented here provide the first evidence of a T6SS effector protein that affects the physiology of F. tularensis, namely, the growth, cell size, viability, and aminoglycoside resistance of F. tularensis LVS. This study also adds insight into our understanding of OpiA as a determinant of virulence. Finally, the fluorescence fusion constructs presented here will be useful tools for dissecting the role of OpiA in infection.

A mechanism for FtsZ-independent proliferation in Streptomyces.

The central player in bacterial cell division, FtsZ, is essential in almost all organisms in which it has been tested, with the most notable exception being Streptomyces. Streptomycetes differ from many bacteria in growing from the cell tip and undergoing branching, similar to filamentous fungi. Here we show that limited cell damage, either mechanical or enzymatic, leads to near complete destruction of mycelial microcolonies of a Streptomyces venezuelae ftsZ mutant. This result is consistent with a lack of ftsZ-dependent cross-walls and may be inconsistent with a recently proposed role for membrane structures in the proliferation of ftsZ mutants in other Streptomyces species. Rare surviving fragments of mycelium, usually around branches, appear to be the preferred sites of resealing. Restoration of growth in hyphal fragments of both wild-type and ftsZ mutant hyphae can occur at multiple sites, via branch-like outgrowths containing DivIVA protein at their tips. Thus, our results highlight branching as a means of FtsZ-independent cell proliferation.

Dynamic gradients of an intermediate filament-like cytoskeleton are recruited by a polarity landmark during apical growth.

Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces.

In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes.

Domains involved in the in vivo function and oligomerization of apical growth determinant DivIVA in Streptomyces coelicolor.

The coiled-coil protein DivIVA is a determinant of apical growth and hyphal branching in Streptomyces coelicolor. We have investigated the properties of this protein and the involvement of different domains in its essential function and subcellular targeting. In S. coelicolor cell extracts, DivIVA was present as large oligomeric complexes that were not strongly membrane associated. The purified protein could self-assemble into extensive protein filaments in vitro. Two large and conspicuous segments in the amino acid sequence of streptomycete DivIVAs not present in other homologs, an internal PQG-rich segment and a carboxy-terminal extension, are shown to be dispensable for the essential function in S. coelicolor. Instead, the highly conserved amino-terminal of 22 amino acids was required and affected establishment of new DivIVA foci and hyphal branches, and an essential coiled-coil domain affected oligomerization of the protein.