Corrigendum: An Immunomodulatory Transcriptional Signature Associated With Persistent Listeria Infection in Hepatocytes.

[This corrects the article DOI: 10.3389/fcimb.2021.761945.].

The unforeseen intracellular lifestyle of Enterococcus faecalis in hepatocytes.

Enterococcus faecalis is a bacterial species present at a subdominant level in the human gut microbiota. This commensal turns into an opportunistic pathogen under specific conditions involving dysbiosis and host immune deficiency. E. faecalis is one of the rare pathobionts identified to date as contributing to liver damage in alcoholic liver disease. We have previously observed that E. faecalis is internalized in hepatocytes. Here, the survival and fate of E. faecalis was examined in hepatocytes, the main epithelial cell type in the liver. Although referred to as an extracellular pathogen, we demonstrate that E. faecalis is able to survive and divide in hepatocytes, and form intracellular clusters in two distinct hepatocyte cell lines, in primary mouse hepatocytes, as well as in vivo. This novel process extends to kidney cells. Unraveling the intracellular lifestyle of E. faecalis, our findings contribute to the understanding of pathobiont-driven diseases.

An Immunomodulatory Transcriptional Signature Associated With Persistent Listeria Infection in Hepatocytes.

Listeria monocytogenes causes severe foodborne illness in pregnant women and immunocompromised individuals. After the intestinal phase of infection, the liver plays a central role in the clearance of this pathogen through its important functions in immunity. However, recent evidence suggests that during long-term infection of hepatocytes, a subpopulation of Listeria may escape eradication by entering a persistence phase in intracellular vacuoles. Here, we examine whether this long-term infection alters hepatocyte defense pathways, which may be instrumental for bacterial persistence. We first optimized cell models of persistent infection in human hepatocyte cell lines HepG2 and Huh7 and primary mouse hepatocytes (PMH). In these cells, Listeria efficiently entered the persistence phase after three days of infection, while inducing a potent interferon response, of type I in PMH and type III in HepG2, while Huh7 remained unresponsive. RNA-sequencing analysis identified a common signature of long-term Listeria infection characterized by the overexpression of a set of genes involved in antiviral immunity and the under-expression of many acute phase protein (APP) genes, particularly involved in the complement and coagulation systems. Infection also altered the expression of cholesterol metabolism-associated genes in HepG2 and Huh7 cells. The decrease in APP transcripts was correlated with lower protein abundance in the secretome of infected cells, as shown by proteomics, and also occurred in the presence of APP inducers (IL-6 or IL-1ß). Collectively, these results reveal that long-term infection with Listeria profoundly deregulates the innate immune functions of hepatocytes, which could generate an environment favorable to the establishment of persistent infection.

Commensal bacteria augment Staphylococcus aureus infection by inactivation of phagocyte-derived reactive oxygen species.

Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection.

NAD+ pool depletion as a signal for the Rex regulon involved in Streptococcus agalactiae virulence.

In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host.

Do Primocolonizing Bacteria Enable Bacteroides thetaiotaomicron Intestinal Colonization Independently of the Capacity To Consume Oxygen?

Aerobic bacteria are frequent primocolonizers of the human naive intestine. Their generally accepted role is to eliminate oxygen, which would allow colonization by anaerobes that subsequently dominate bacterial gut populations. In this hypothesis-based study, we revisited this dogma experimentally in a germfree mouse model as a mimic of the germfree newborn. We varied conditions leading to the establishment of the dominant intestinal anaerobe Bacteroides thetaiotaomicron Two variables were introduced: Bacteroides inoculum size and preestablishment by bacteria capable or not of consuming oxygen. High Bacteroides inoculum size enabled its primocolonization. At low inocula, we show that bacterial preestablishment was decisive for subsequent Bacteroides colonization. However, even non-oxygen-respiring bacteria, a hemAEscherichia coli mutant and the intestinal obligate anaerobe Clostridium scindens, facilitated Bacteroides establishment. These findings, which are supported by recent reports, revise the long-held assumption that oxygen scavenging is the main role for aerobic primocolonizing bacteria. Instead, we suggest that better survival of aerobic bacteria ex vivo during vectorization between hosts could be a reason for their frequent primocolonization.

Intestinal translocation of enterococci requires a threshold level of enterococcal overgrowth in the lumen.

Enterococci are subdominant members of the human gastrointestinal microbiota. Enterococcus faecalis is generally harmless for healthy individuals, but it can cause a diverse range of infections in immunodeficient or elderly patients with severe underlying diseases. In this study, we analysed the levels of intestinal translocation of indigenous enterococci in C57BL/6, CF-1 and CX3CR1-/- mice upon clindamycin antibiotic-induced dysbiosis. We found that C57BL/6 was the most permissive model for enterococcal translocation and that initiation of E. faecalis translocation coincided with a threshold of enterococcal colonisation in the gut lumen, which once reached, triggered E. faecalis dissemination to deeper organs. We showed that the extent to which E. faecalis clinical strain VE14821 competed with indigenous enterococci differed between the C57BL/6 and CX3CR1-/- models. Finally, using a simplified gnotobiotic model, we observed E. faecalis crossing an intact intestinal tract using intestinal epithelial cells as one route to reach the lamina propria. Our study opens new perspectives for assessing the effect of various immunodeficiencies and for investigating mechanisms underlying enterococcal translocation.

Exploration of the role of the virulence factor ElrA during Enterococcus faecalis cell infection.

Enterococcus faecalis, an organism generally not pathogenic for healthy humans, has the potential to cause disease in susceptible hosts. While it seems to be equipped to interact with and circumvent host immune defense, most of the molecular and cellular mechanisms underlying the enterococcal infectious process remain elusive. Here, we investigated the role of the Enterococcal Leucine Rich protein A (ElrA), an internalin-like protein of E. faecalis also known as a virulence factor. ElrA was previously shown to prevent adhesion to macrophages. We show that ElrA does not inhibit the basic phagocytic process, but is able to prevent sensing and migration of macrophages toward E. faecalis. Presence or absence of FHL2, a eukaryotic partner of ElrA, does not affect the ElrA-dependent mechanism preventing macrophage migration. However, we highlight a partial contribution of FHL2 in ElrA-mediated virulence in vivo. Our results indicate that ElrA plays at least a dual role of which anti-phagocytic activity may contribute to dissemination of extracellular E. faecalis during infection.

Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae.

Heme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection.

A partial metabolic pathway enables group b streptococcus to overcome quinone deficiency in a host bacterial community.

Aerobic respiration metabolism in Group B Streptococcus (GBS) is activated by exogenous heme and menaquinone. This capacity enhances resistance of GBS to acid and oxidative stress and improves its survival. In this work, we discovered that GBS is able to respire in the presence of heme and 1,4-dihydroxy-2-naphthoic acid (DHNA). DHNA is a biosynthetic precursor of demethylmenaquinone (DMK) in many bacterial species. A GBS gene (gbs1789) encodes a homolog of the MenA 1,4-dihydroxy-2-naphthoate prenyltransferase enzyme, involved in the synthesis of demethylmenaquinone. In this study, we showed that gbs1789 is involved in the biosynthesis of long-chain demethylmenaquinones (DMK-10). The Δgbs1789 mutant cannot respire in the presence of heme and DHNA, indicating that endogenously synthesized DMKs are cofactors of the GBS respiratory chain. We also found that isoprenoid side chains from GBS DMKs are produced by the protein encoded by the gbs1783 gene, since this gene can complement an Escherichia coli ispB mutant defective for isoprenoids chain synthesis. In the gut or vaginal microbiote, where interspecies metabolite exchanges occur, this partial DMK biosynthetic pathway can be important for GBS respiration and survival in different niches.

HrtBA and menaquinones control haem homeostasis in Lactococcus lactis.

Lactococcus lactis is a fermenting Gram-positive bacterium widely used for production of dairy products. Lacking haem biosynthesis genes, L. lactis can still shift to an energetically favourable respiratory metabolism by activating a terminal cytochrome bd oxidase when haem is added to an aerated culture. Haem intracellular homeostasis is mediated by the hrtRBA operon encoding the conserved membrane HrtBA haem efflux permease and the unique intracellular haem sensor and regulator, HrtR. Here we report that membrane-associated menaquinones (MK) favour the accumulation of reduced haem in membranes. An oxidative environment, provided by oxygen, prevents and reverses haemin reduction by MK and thus limits haem accumulation in membranes. HrtBA counteracts MK-dependent membrane retention of excess haem in membrane, suggesting direct efflux from this compartment. Moreover, both HrtBA and MK-mediated reduction have a strong impact on haem intracellular pools, as determined via HrtR haem sensor induction, suggesting that intracellular haem acquisition is controlled at the membrane level without the need for dedicated import systems. Our conclusions lead to a new hypothesis of haem acquisition and regulation in which HrtBA and the bacterial membrane have central roles in L. lactis.

Nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae, attacks the neutrophil extracellular traps and is needed for full virulence.

Most bacteria of the genus Streptococcus are opportunistic pathogens, and some of them produce extracellular DNases, which may be important for virulence. Genome analyses of Streptococcus agalactiae (GBS) neonate isolate NEM316 revealed the presence of seven genes putatively encoding secreted DNases, although their functions, if any, are unknown. In this study, we observed that respiration growth of GBS led to the extracellular accumulation of a putative nuclease, identified as being encoded by the gbs0661 gene. When overproduced in Lactococcus lactis, the protein was found to be a divalent cation-requiring, pH-stable and heat-stable nuclease that we named Nuclease A (NucA). Substitution of the histidine(148) by alanine reduced nuclease activity of the GBS wild-type strain, indicating that NucA is the major nuclease ex vivo. We determined that GBS is able to degrade the DNA matrix comprising the neutrophil extracellular trap (NET). The nucA(H148A) mutant was impaired for this function, implicating NucA in the virulence of GBS. In vivo infection studies confirmed that NucA is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that NucA is involved in NET escape and is needed for full virulence.

The Staphylococcus aureus Opp1 ABC transporter imports nickel and cobalt in zinc-depleted conditions and contributes to virulence.

Metals are common enzymatic cofactors, and their acquisition must be assured under the various conditions encountered in the host. Although some strategies for acquisition of common metals such as iron and manganese have been elucidated, little is known about the conditions and mechanisms used to capture trace metals. Nickel is a transition metal required as a cofactor for several bacterial enzymes, including urease. Staphylococcus aureus does express a nickel ABC transporter, Nik, which functions in metal-replete medium and is necessary for nickel urease activity and urinary tract colonization. In this work, we identified a novel cobalt and nickel transporter, which we named Cnt (previously annotated Opp1), in the major opportunistic pathogen S. aureus. Metal transport activity was revealed by growing cells in a chemically defined medium devoid of metals. Zinc specifically inhibits Cnt-mediated nickel and cobalt uptake, on both functional and transcriptional levels. Mortality due to S. aureus cnt mutant in systemic infection and colonization of the bladder and kidneys in ascending urinary tract infection model were reduced compared to the parent strain. This study identifies a novel S. aureus trace metal transporter and its restricted conditions of activity, and establishes its role in infection.

Two coregulated efflux transporters modulate intracellular heme and protoporphyrin IX availability in Streptococcus agalactiae.

Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for 'porphyrin-regulated efflux'. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The DeltapefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host.