Active modification of host inflammation by Salmonella

Abstract

The dampening of host immune responses is a critical aspect of pathogenesis for the enteropathogen Salmonella enterica. Our laboratory has recently described a role for the secreted effector GogB in disruption of NFκB activation and limitation of the host inflammatory response to infection. GogB alters the NFκB pathway by preventing IκB degradation by the host SCF E3 ubiquitin ligase, through an interaction with Skp1 and FBXO22. The prevention of NFκB activation through this interaction dampens the host inflammatory response in the gut, which in turn limits the damage to host tissues during chronic infection. In this addendum, we summarize these recent findings and discuss their implications and impact in the area of host-pathogen interactions.

Introduction

Host-pathogen interactions involve interplay between host immune responses and the molecular armory of the pathogen. For bacterial pathogens, maximizing survival and replication while minimizing host immune activation is a careful balancing act that seeks to avoid an uncontrolled host inflammatory response.¹ The human enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) achieves this balance through delivery of pro- and anti-inflammatory effector proteins to the host cell by means of a syringe-like type III secretion system (T3SS). Two T3SS are encoded within the Salmonella Pathogenicity Islands (SPI-) 1 and 2 and function in host cell invasion, survival and systemic dissemination.^2-4 Whereas several effectors enhance bacterial uptake and tissue colonization by eliciting host inflammation at the early stages of infection, a subset of effectors dampen the host immune response by blocking inflammation resulting in a mechanism by which the bacteria control their population density and pathological impact on the host.^1,5-8

In recent work published in PLoS Pathogens,⁹ we investigated the function of GogB, an example of one such anti-inflammatory effector delivered to host cells by S. Typhimurium. We previously showed that GogB is secreted by both the SPI-1 and SPI-2 T3SS and has sequence similarity to a group of effectors that function as novel E3 ubiquitin ligases (NELs) however its mechanism of action and cellular target remained hitherto unknown.¹⁰ Our most recent findings revealed that GogB functions to limit tissue damage from enhanced inflammatory host response during S. Typhimurium infection. We showed that GogB interacts with the host proteins Skp1 and the F-box protein FBXO22 of the E3 SCF (Skp1-Cullin1-F-box) ubiquitin ligase to inhibit the transcription factor NFκB (nuclear factor kappa B) from activating pro-inflammatory gene expression. In this addendum, we provide additional insights into the mechanism of GogB interaction with the host ubiquitination system and its role in dampening intestinal inflammation in NRAMP^+/+ mice.

GogB Interacts with the Host Ubiquitination System

The highly conserved eukaryotic ubiquitination system is involved in cellular processes such as cell cycle regulation, signal transduction and control of host immunity.¹¹ Ubiquitination is the reversible post-translational modification of a protein by addition of ubiquitin molecules that target it for proteosomal degradation, or that change its subcellular localization. The absence of ubiquitination in bacteria makes this system a common target of pathogens to subvert host physiology and modulate immune response.^12-15 In a case of molecular mimicry, a group of effectors called NELs found in Salmonella, Shigella and Yersinia species functions as ubiquitin ligases. The effectors are distinguished by N-terminal leucine-rich repeats (LRR) and a C-terminal catalytic cysteine residue that mediates ubiquitination.^16,17 While the NEL ligases could be targeting the host inflammatory pathway an in vivo role of the ubiquitin ligase activity remains to be elucidated.¹⁸ Based on a previous analysis of GogB that showed an N-terminal LRR region, we hypothesized that GogB manipulates the host ubiquitination machinery. Using a proteomic approach we identified an interaction between GogB and the human F-box protein, FBXO22, which we verified in epithelial and macrophage cell culture. As components of the E3-type SCF ligase, F-box proteins bind to the adaptor protein Skp1 (S-phase kinase-associated protein 1) and recognize substrates for ubiquitination.¹⁹ We also showed that GogB was associated with Skp1 following delivery by S. Typhimurium into infected macrophages. Functional deletion experiments found that the GogB C-terminal region mediates binding to FBXO22 and Skp1. F-box proteins contain a ~50 amino acid F-box motif at the N-terminus and C-terminal WD40 repeats or LRRs.¹⁹ Similarly, we found an F-box like motif within the GogB C-terminal portion containing conserved leucine and proline residues considered essential for F-box-Skp1 complex formation in eukaryotes.²⁰ Deletion of the F-box like domain (but not mutation of the individual Leu or Pro residues) severely reduced binding to Skp1, indicating that additional residues in the F-box like domain are critical for the Skp1-GogB interaction.

The presence of a GogB-FBXO22-Skp1 complex suggested the potential for distinct binding sites on GogB for host proteins (Fig. 1). To determine whether Skp1 was required for GogB-FBXO22 binding, we knocked down Skp1 expression in HeLa cells and infected these cells with S. Typhimurium. Pull-down assays showed GogB co-purifying with FBXO22 in the absence of Skp1. Crystal structures of Skp1 and the human F-box protein Skp2 show extensive binding interfaces in which the F-box motif and part of the LRRs in Skp2 interdigitate with the N-terminal BTB/POZ fold and C-terminal extension of Skp1.²⁰ Whether GogB binds directly to Skp1 through a similar interface or requires FBOX22 for GogB-Skp1 complex formation remains to be determined. Studying the dynamics of the GogB and SCF ligase interaction would present a structural view of how a bacterial effector binds protein targets to modulate the host ubiquitination machinery.

Figure 1. The S. Typhimurium effector GogB interacts with SCF (Skp1-Cullin1-F-box) ubiquitin ligase complex. GogB has N-terminal leucine-rich repeats (LRRs) similar to a group of effectors called novel E3 ligases (NELs) found in Salmonella, Shigella and Yersinia. The GogB C-terminus contains an F-box-like domain that mediates binding to the F-box protein FBXO22 and the adaptor protein Skp1. A model of the GogB-FBXO22-Skp1 complex shows the N-terminal BTB/POZ fold and a C-terminal extension of Skp1 as putative interfaces for binding the F-box motif on FBXO22 and the GogB C-terminus.

In western blot analyses of infected cell lysates, we have not observed higher molecular weight bands that could indicate that GogB is modified in the host cell by ubiquitination. Demonstration of a ubiquitinase activity by GogB and identification of a catalytic cysteine residue would indicate whether it functions similar to the NEL ligases. However, our initial attempts to test GogB in an in vitro ubiquitinase assay were hampered by the instability of recombinant GogB. As such, alternative methods to purify a stable form of GogB for further biochemical analyses are warranted. Examination of the C-terminal domain of GogB reveals cysteine residues at position 401 and within the F-box domain (264–352) but whether these residues are required for GogB function will be the subject of future studies.

GogB: Part of a Molecular Rheostat for Host Inflammation?

NF-κB is a transcription factor responsible for homeostasis in cells of the intestinal tract as well as early detection of pathogens. Under naive conditions, NFκB typically exists in its inactive form in the cytosol bound to the inhibitory protein, IκB. Upon stimulation of membrane bound or cytoplasmic pattern recognition receptors (PRRs), the IκB kinase (IKK) is activated and phosphorylates IκB causing its dissociation from NFκB. Once dissociated, IκB is marked for proteosomal degradation through ubiquitination by the SCF^βTRcP1 ligase,²¹ allowing NFκB to traverse the nuclear membrane and activate expression of target genes (Fig. 2). Because several Salmonella effectors target the NFκB pathway, we asked whether the interaction of GogB with the SCF ligase plays a role in inflammation.^22,23 Our results showed that macrophage-like cells infected with gogB-deficient S. Typhimurium had lower amounts of IκBα compared with infection with wild type bacteria and that treatment of infected cells with the proteosome inhibitor MG-132 lead to accumulation of poly-ubiquitinated IκBα in ΔgogB-infected cells. Luciferase reporter assays showed higher NFκB and interleukin 1β (IL1β) levels in cells infected with ΔgogB and gogB mutants lacking the C-terminal region or F-box like domain compared with wild type S. Typhimurium. These effects were abolished after knockdown of Skp1 expression in host cells, denoting that Skp1 is required for GogB-mediated inhibition of IκBα poly-ubiquitination and degradation (Fig. 2).

Figure 2. GogB blocks the NFκB pathway to suppress inflammation. In the inactive state, NFκB is bound by its inhibitor IκB in the cytoplasm. In the presence of a signal, IκB is phosphorylated by IKK and is then ubiquitinated by the SCF ligase complex to target IκB for proteosomal degradation. The dissociation of IκB allows NFκB to enter the nucleus and induce pro-inflammatory gene expression. During infection, GogB interacts with the F-box protein FBXO22 and Skp1 of the SCF ligase to inhibit poly-ubiquitination of IκB, which blocks NFκB activation and dampens inflammation.

The GogB-SCF ligase interaction in dampening host inflammatory response implies that GogB could be acting as a molecular mimic of F-box proteins similar to virulence factors found in the pathogens Legionella, Ralstonia solanacearum and poxviruses.^12-15 Skp1 stabilizes the conformation of F-box proteins so the binding of GogB to Skp1 could be required for its proper function.²⁴ Since Skp1 complexes with diverse F-box proteins, it is also possible that GogB blocks the recruitment of F-box proteins such as βTRcP1 to the SCF ligase, thereby interfering with the NFκB pathway.

The function of FBXO22 is not fully understood but a recent report has implicated its role in KDM4A histone demethylase turnover, a process that takes place in the nucleus.²⁵

Polyubiquitination and demethylation are mutual processes that regulate gene expression. The amount of demethylases is controlled by proteosomal degradation to regulate their activity and levels of expression of target genes. Demethylases have been implicated in inflammation in response to bacterial LPS and regulation of chemokine expression.^26,27 Further work on the GogB-FBXO22 interaction would indicate a potential role of a Salmonella effector in regulating demethylases to control inflammation during infection.

Interestingly, we only observed the GogB-FBXO22 complex when GogB was delivered by the S. Typhimurium T3SS into host cells. This suggests that GogB may require a co-delivered T3SS effector for its function. Whether GogB interacts with NELs or other effectors targeting the NFκB pathway such as AvrA^22,28 and SseL²³ represents an intriguing line of investigation. The potential for GogB to recognize and act on pro-inflammatory activities generated by other translocated effectors may be an additional level of regulation for effector function within the host cell.²⁹

The Beneficial Effects of GogB on Limiting Host Tissue Damage

NFκB has been associated with maintenance of homeostasis within the gut but has also been implicated in driving colitis.^30-32 Thus tight regulation of the activation and suppression of NFκB activity is critical even within the native state of the host. Upon infection, activation of NFκB and the resulting transcription of pro-inflammatory molecules are the convergent downstream event from multiple PRRs in intestinal epithelial cells.²¹ Pathogen recognition and initiation of these early responses are critical for establishing a robust immune response, eventually enabling clearance of the infection. However, uncontrolled inflammation can result in undesirable bystander damage toward the host. In the gut, this is typically described as colitis. S. Typhimurium is capable of inducing enterocolitis in both cattle and humans, but mice are typically resistant to developing similar levels of inflammation within the intestine as a result of infection.^33,34 This innate resistance in mice can be disrupted through perturbation of the murine microbiota with administration of an oral antibiotic prior to infection. The use of streptomycin is well known for provoking colitis in a Salmonella infection model.³⁵ Previous work investigating the role of S. Typhimurium effectors SseL and AvrA, both of which dampen the NFκB response, were performed in C57BL/6 (B6) mice with antecedent streptomycin treatment and attenuated mutants or sub-lethal dose of bacteria.^22,23 In the absence of streptomycin pretreatment, our investigation did not yield an observable phenotype for gogB mutants in B6 mice in a competitive infection with wild type S. Typhimurium. To address the issue that during co-infection the native GogB produced by wild type bacteria could be negating the effect of gogB deletion on inflammation, we later performed infections of B6 mice with either ΔgogB or wild type S. Typhimurium. However, these experiments yielded no observable differences in organ bacterial loads at day 4 post infection, nor any changes in gross pathology (unpublished data). Whether use of streptomycin prior to infection would allow us to find a phenotype in B6 mice remains to be determined. However, the lack of a phenotype in this acute model of infection lead us to question whether GogB may play a role during chronic bacterial infection to dampen the host immune response and support long-term carriage.

Chronically infected mouse models of S. Typhimurium infection are important for our understanding of not only transmission of infection but also the machinery required for an organism to persist without killing its host or inducing high degrees of inflammation.^36,37 Whether a mouse strain can be used for a chronic infection with wild type S. Typhimurium is dependent upon the presence of functional Nramp1. Nramp1 (Natural resistance associate macrophage protein 1), also called Slc11A1,³⁸ is a protein located in the membrane of lysogenic vesicles in macrophages and dendritic cells that affects the degree of pro-inflammatory responses and limits the access of bacteria residing within these vesicles to divalent cations, which are critical for their replication.^39-42 Strains of mice designated NRAMP^−/− have a single mutation at amino acid 169, which leads to improper protein folding and therefore loss of protein stability.⁴³ Mice that carry this mutation are highly susceptible to S. Typhimurium infection, eventually succumbing to infection within a week.³³ To address the hypothesis that dampening of NF-κB responses aid in long-term carriage, we examined the role of GogB in a long-term, chronic infection of Salmonella-resistant (NRAMP^+/+) mice. We found that infection of NRAMP^+/+ Sv129 mice with gogB-deficient S. Typhimurium produced amplified immune responses that were accompanied by increased numbers of bacteria in the cecal tissue (Fig. 3) despite greater infiltration of the cecum by neutrophils and monocytes, as well as lymphocytes. While increased levels of inflammation drastically affected bacterial numbers in the intestines of NRAMP^+/+ mice, bacterial loads at systemic sites were comparable between bacteria with or without GogB. This could be due to the different mechanisms of host invasion utilized by S. Typhimurium at systemic vs. enteric sites. In the gut, S. Typhimurium exists extracellularly as well as within epithelial cells and the underlying lamina propria, allowing for its continuing activation of the host. Systemically, S. Typhimurium persists mainly within Salmonella-containing vesicles (SCVs) in macrophages.⁴⁴ Bacteria in SCVs are sequestered away from PRRs, preventing NFκB activation and limiting the effect of the loss of GogB on bacterial numbers at these sites. Histological analysis of infected systemic organs in order to detect increased inflammatory responses was not performed during our investigations but would be of interest in order to compare the phenotypic effect of GogB deletion with other effectors targeting NFκB activation, as well as address why increased systemic inflammation has limited to no effect on bacterial load at these sites.

Figure 3. Schematic diagram of cecal inflammation in the presence and absence of GogB. In the absence of streptomycin pretreatment, Salmonella infection results in limited inflammatory responses within the gut. However, within four days following ΔgogB Salmonella infection of NRAMP^+/+ mice, vastly increased levels of inflammatory cell infiltrates are present in the cecal tissue, as well as increased transcript levels of pro-inflammatory chemokines and cytokines. The effect of GogB modulation of NFκB activation plays a critical role in tempering inflammation in the gut in response to infection.

With both increased bacterial levels and increased inflammation present at intestinal sites, it was unclear whether one phenomenon preceded the other, or whether GogB itself could affect bacterial replication at this site. It was recently shown that Salmonella utilizes intestinal inflammation to out-compete the indigenous microbiota of the host.⁴⁵ Salmonella outgrowth during intestinal inflammation has been attributed to its ability to use tetrathionate produced in the gut in the presence of reactive oxygen species generated during an inflammatory response.⁴⁶ Treatment of NRAMP^+/+ mice with the anti-inflammatory steroid dexamethasone, resulted in equivalent numbers of wild type and ΔgogB mutant bacteria in the gut, suggesting that the inflammation resulting from infection with the ΔgogB strain was directly related to the bacterial outgrowth. Thus, our work offers additional evidence that inflammation precedes bacterial outgrowth in the gut of NRAMP^+/+ mice.

That we were only able to observe an effect of deletion of gogB in NRAMP^+/+ mice is an interesting and open question. Recent studies have shown that the insertion of a neomycin cassette within the NRAMP gene to disrupt Nramp1 function in 129 mice resulted in significantly decreased inflammatory responses to S. Typhimurium infection even within the first 24 h post infection.⁴⁷ This implies that the Nramp1 protein has a direct effect on inflammatory responses, which the authors postulated could be due to increased monocyte and neutrophil responses.^47,48 Future investigations into the cellular sources of inflammation observed in NRAMP^+/+ mice in response to ΔgogB S. Typhimurium infection is warranted, as well as whether these responses are dependent upon NRAMP status. Such studies are likely to yield new insight into the role of host genetics in the outcome of host-pathogen interactions.

Glossary

Abbreviations:

T3SS

type III secretion system

SCF

Skp1-Cullin-F-box

NFκB

nuclear factor kappa B

IκBα

inhibitor of kappa B alpha

NEL

novel E3 ligase

LRR

leucine-rich repeat

Pilar AV, Reid-Yu SA, Cooper CA, Mulder DT, Coombes BK. GogB is an anti-inflammatory effector that limits tissue damage during Salmonella infection through interaction with human FBXO22 and Skp1. PLoS Pathog. 2012;8:e1002773. doi: 10.1371/journal.ppat.1002773.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.