An Innate Pathogen Sensing Strategy Involving Ubiquitination Of Bacterial Surface Proteins Part 2

DISCUSSION

Metazoans use ubiquitination as a versatile mechanism to maintain cytosolic homeostasis, preventing the accumulation of damaged proteins/organelles and defending against invading pathogens (40, 41). Given the variety of pathogens and diverse sets of damaged proteins encountered, the identification of common motifs for substrate recognition and subsequent ubiquitination represents a smart strategy for resource optimization. Proteins in eukaryotic cells that are destined for proteolysis are typically identified by a tripartite degron motif (24). 

Immunity is the body's ability to fight off disease, including responses to bacteria, viruses, and other harmful substances. If the body's immune system is compromised, it will not be strong enough to deal with the onslaught of disease. Studies have shown that malnutrition has an impact on the function of the immune system, making the body more susceptible to infection and disease. Protein is one of the important substances to maintain immunity.

Proteins are made of amino acids, some of which are called essential amino acids because they must be provided by food and cannot be synthesized by the body. These essential amino acids include tryptophan, leucine, phenylalanine, lysine, isoleucine, methionine, valine, and histidine. If the body does not get enough essential amino acids, it can lead to impaired protein metabolism, which can affect the function of the immune system.

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This prompted us to explore whether similar motifs could be found on pathogen surfaces, where they might function as a proxy for substrate recognition. Here, we demonstrate that host ubiquitin ligases use similar molecular signatures to sense phylogenetically distinct pathogens. Pathogen recognition via common molecular patterns/motifs (PAMPs) is a well-characterized feature of innate immunity (42). Our results suggest that degron-like sequences within bacterial surface proteins act in a manner equivalent to PAMPs, to impart intracellular pathogen surveillance. This modus operand could be further exploited by the host for the presentation of microbial protein antigens on major histocompatibility complex I to induce a strong CD8 response directed against intracellular pathogens. 

The potential importance of ubiquitin-mediated detection of pathogens to host defenses is further highlighted by the substantial residual ubiquitination detected in mutant SPN that lack both BgaA and PspA. This indicates that there are further, unidentified substrates of the ubiquitin machinery, with the resulting redundancy ensuring robust pathogen interception. Notably, some immediate questions about the degron motif and its importance to the bacteria (other than being a recognizable unit) provide an interesting evolutionary angle. 

The degron motif is dispensable in vivo, and if anything, mutating or deleting the motif improved intracellular persistence resulting in increased virulence. This is substantiated by our results that mutation in the degron motif could be used by pathogens to escape ubiquitin-mediated recognition as seen for SPN serotype 19F. However, the conserved nature of the degron motif in BgaA across the majority of the SPN serotypes could be suggestive of a counter strategy adopted by bacteria to be less lethal to the host. 

This could provide an opportunity for the pathogen to increase its occupiable habitats. Concurrently, ubiquitination of the bacterial surface or secreted proteins at the degron motif could modulate them functionally to dampen or rewire the host response for eventual benefit (43). Therefore, the presence or absence of eukaryotic-like functional domains or motifs (such as degrons) could be dependent on a pathogenic niche and adapted to evade or modulate host immune responses.

A similar pathogen sensing mechanism involving guanylate binding proteins has also been implicated in bacterial clearance; however, unlike ubiquitination, their role is reported to be restricted to Gram-negative pathogens (44–47). Ubiquitination targets the pathogen irrespective of Gram origin, for instance, RNF213 has been documented to target Listeria spp. irrespective of the absence of LPS (48). We, therefore, do not eliminate the possibility of its antimicrobial action against SPN too. 

A pathogenic surface can be ubiquitinated with multiple chain types by different E3 ligases with interchain interactions (41). In particular, in the case of Mtb, Smurf1, and Parkin have been proven to form K48 and K63 chain types that function synergistically to degrade the pathogen (12). Understanding the mechanistic role of K48 ubiquitination and the E3 ligases involved during infection scenarios is still understudied. In the case of STm, a single E3 ligase ARIH1 has been depicted to target cytosolic STm with K48 ubiquitin chains leading to their elimination from the system (15). Based on our and others’ findings, K48-Ub–labeled pathogens are lastly degraded while being associated with proteasomal machinery. 

However, this study shows K48-Ub decoration of specific bacterial surface proteins. Some pathogens are known to actively modify their surface proteins, protecting them from ubiquitination and subsequent killing, thus emphasizing the significance of surface localization of the ubiquitin substrate (10). In addition, multiple obligate intracellular pathogens have evolved strategies to de-ubiquitinate themselves, or host regulatory components, to evade ubiquitin-mediated clearance (5). Together, these findings underscore the importance of ubiquitin-mediated alarm arousal as a fundamental intracellular pathogen sensing mechanism central to host defenses.

Our study further demonstrated the central role of SCF E3 ligase, particularly with FBXW7, in bacterial ubiquitination augmenting pathogen degradation. Heterozygous mutations in FBXW7, particularly R505C (a critical residue in substrate recognition pocket) variant, trigger multiple carcinomas and lymphocytic leukemia in humans (49, 50). A recent cohort-based study indicated that almost 43% of the patients with chronic lymphocytic leukemia (CLL) succumb to bacterial pneumonia and sepsis, followed by fungal infections (51). 

This corroborates our observation of the abrogated ability of host cells bearing FBXW7 mutation to sense and eliminate pathogens. Our findings, therefore, provide an unexpected molecular explanation of the enhanced risk of infections in patients with CLL. The link between genetic polymorphism in E3 ligase genes and susceptibility to bacterial infections is also substantiated by patients with Parkinson’s disease, who are vulnerable to typhoid fever or leprosy (14), suggesting the noteworthy contribution of E3 ligases in host immunity against bacterial infections and maintenance of cellular steady state. 

In conclusion, we deciphered a universal language of sensing cytosol-dwelling pathogens that could be efficiently applied by the host to recognize microorganisms for subsequent elimination. Collectively, these findings shed light on understanding the rudimentary cellular immune processes, which could be harnessed to intensify antibacterial immunity.

MATERIALS AND METHODS

Cell culture

Both the human lung alveolar carcinoma (type II pneumocyte) cell line A549 [American Type Culture Collection (ATCC) no. CCL185)] and the cervical adenocarcinoma cell line HeLa (ATCC no. CRM-CCL-2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HiMedia) supplemented with 10% fetal bovine serum (Gibco) at 37°C and 5% CO2.

Bacterial strains and growth conditions

SPN (R6, serotype 2, gift from Tim J. Mitchell, University of Birmingham, United Kingdom) was grown in Todd-Hewitt broth supplemented with 1.5% yeast extract at 37°C in 5% CO2. The following antibiotics were used to grow SPN cultures when required: kanamycin (200 μg/ml), spectinomycin (100 μg/ml), and chloramphenicol (4.5 μg/ml). Nine hundred microliters of 0.4 OD600 (optical density at 600 nm) grown SPN culture were mixed with 600 μl of 80% sterile glycerol (32% final glycerol concentration) and stored in a −80°C deep freezer. These glycerol stocks were used as starting inoculum for all experiments.

Escherichia coli DH5α and STm (ATCC 14028) cultures were grown in Luria-Bertani broth (LB) at 37°C under shaking conditions (200 rpm), and when necessary, the following antibiotics were used: kanamycin (50 μg/ml), spectinomycin (100 μg/ml), chloramphenicol (20 μg/ml), and ampicillin (100 μg/ml). For all infection assays, bacteria were grown till OD600 ~0.4, followed by resuspension in phosphate-buffered saline (PBS) to a similar density before infecting the host cells.

Screening of putative ubiquitin target proteins

All surface proteins present in the STm and SPN proteome were first identified from published literature. Complete protein sequences of the surface proteins were obtained from UniProt, which were then used to identify the presence of degron motifs. A set of 29 putative degron motifs were curated from published literature (24, 52, 53) and the Eukaryotic Linear Motif database. Python’s regex module was used to identify the location of all such curated motifs in the surface protein sequences. A linear search was further performed to identify the presence of a lysine residue nearby (8 to 14 amino acid residues) to every identified degron motif. This lysine residue is presumed to act as the attachment site for ubiquitin moiety. Last, the shortlisted protein sequences were fed into IUPred (54), a protein structure predicting tool, to locate the presence of a disordered region in between the degron motif and proximal lysine. The resultant proteins (BgaA and PspA for SPN and RlpA for STm) were selected for evaluation as ubiquitination targets and decoding of their role in pathogen clearance.

Bacterial strain construction

Allelic exchange by homologous recombination was carried out using gene flanking regions carrying an inserted antibiotic cassette for the generation of mutant strains in both SPN and STm (table S2).

For SPN, 500-bp upstream and downstream regions of bgaA, pspA, and his genes were amplified from the genome with appropriate primers (table S3) and assembled in the pBKS vector. Following cloning of these fragments, an antibiotic resistance cassette (spectinomycin for bag as well as pspA and chloramphenicol for hysA) was inserted into this construct. The linearized recombinant plasmids were then transformed into WT SPN using competence stimulating peptide 1 (GenPro Biotech), recombinants were selected using respective antibiotics, and gene replacement was confirmed by polymerase chain reaction (PCR) and sequencing of the respective gene loci. For STm, the λ-red recombinase method was used for the generation of gene deletion mutants (55). 

Briefly, sequences homologous to the ends of the rope gene were appended in the primer sequences that are used for the amplification of the kanamycin resistance cassette. The cassette was then electroporated into the WT STm strain, and knockouts generated by homologous recombination were selected based on kanamycin resistance. Gene deletion was confirmed using PCR and sequencing of the gene locus. Fulllength pspA, hysA, and a truncated bgaA-T (1 to 3168 bp) were cloned under the P23 promoter in the shuttle vector pIB166 (56) and used for complementation. 

These recombinant plasmids were also used for site-directed mutagenesis to generate different variants of bgaA-T (bgaA-TK96R, bgaA-TK97R, bgaA-TK96R, K97R, and bgaATΔDegron), pspA (pspAK314R, pspAK315R, pspAK314R, K315R, and pspAΔDegron), and his (hysADegron-BgaA and hysADegron-PspA) using appropriate primer sets (table S3) and transformed into ΔbgaA and ΔpspA mutants. All clones were verified by DNA sequencing. Expression of different variants of BgaA, PspA, and HysA was confirmed by Western blot using appropriate antibodies.

Antibodies and reagents

Anti-Enolase and anti-PspA serum (S. Hammerschmidt, University of Greifswald, Germany); anti-BgaA serum (S. King, The Ohio State University, USA); and antibodies specific for His6 (Invitrogen, MA1-21315), K48-Ub linkage (Millipore, 05-1307), K63-Ub linkage (Millipore, 14-6077-80), Ply (Santa Cruz Biotechnology, sc-80500), FBXW7 (Bethyl Laboratories, A301-721A), SKP1 (Invitrogen, MA5-15928), Cullin1 (Invitrogen, 71-8700), glyceraldehyde phosphate dehydrogenase (Millipore, MAB374), GSK3β [Cell Signaling Technology (CST), D5C5Z], phosphothreonine (CST, 9381S), IgA, Kappa from murine myeloma, clone TEPC-15 (Sigma-Aldrich, M1421), and PSMB7 (Invitrogen, PA5-111404) were procured. The following secondary antibodies were used: horseradish peroxidase (HRP)–tagged anti-rabbit (BioLegend, 406401), HRP-tagged anti-mouse (BioLegend 405306), anti-rabbit Alexa Fluor 488 (Invitrogen, A27206), anti-rabbit Alexa Fluor 555 (Invitrogen, A31572), biotin-conjugated anti-mouse immunoglobulin A (Life Technologies, M31115), anti-mouse Alexa Fluor (Invitrogen, A31570), anti-mouse Alexa Fluor 488 (Invitrogen, A21202), anti-goat Alexa Fluor 633 (Invitrogen, A21082), and FM4-64 (Thermo Fisher Scientific, T13320).

Protein expression and purification

BgaA-T and its variants were cloned in pET28 using Xba I/Not I restriction sites. Recombinant plasmids encoding BgaA-T with N-terminal His-tag were transformed into E. coli BL21 (DE3) cells for protein expression. Freshly transformed colonies were grown in LB containing kanamycin (50 μg/ml) at 37°C on a shaker incubator for 12 hours. One percent of the primary culture was added to 1 liter of LB broth and incubated at 37°C on a shaker incubator till the OD600nm reached between 0.6 and 0.8. Protein expression was induced by the addition of 100 μM isopropyl-β-D-thiogalactopyranoside (IPTG) and growing the culture further at 37°C for 5 to 6 hours with agitation at 150 rpm. 

The cells were harvested by centrifugation at 6000 rpm for 10 min at 4°C. The cell pellet was resuspended in buffer A [25 mM tris (pH 8.0) and 300 mM NaCl] and lysed by sonication. Cell debris was separated by centrifugation (14,000 rpm, 50 min, 4°C), and the supernatant was applied onto a Ni-NTA column, equilibrated with buffer A. The column was washed with 10 column volumes of buffer A, and the His-tagged proteins were eluted with imidazole (250 mM) in buffer A. All the variants of BgaA-T were expressed and purified using the same procedure. FBXW7 was subcloned in the pGEX-4 T-1 vector having an N-terminal glutathione S-transferase (GST) tag from the pCMV6-Entry-FBXW7 vector using the Eco RI restriction enzyme. 

GST-tagged FBXW7 was expressed in E. coli BL21 (DE3) cells following induction of protein expression with 0.1 mM IPTG and growth at 30°C for 4 hours. GST-FBXW7 from E. coli crude extract was purified using Glutathione-Sepharose (GE HealthCare) column chromatography. Fractions containing purified proteins were pooled and concentrated up to 0.5 mg/ml using a 10-kDa molecular weight cutoff filter (Amicon) by centrifugation at 4700 rpm at 4°C. The purity of the proteins was checked on SDS–polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie blue.

Host cell transfections

BgaA-T was cloned in doxycycline-inducible vector pAK_Tol2_TRE_Blast (Addgene no. 130261) using an infusion cloning kit (Takara). A positive clone was confirmed by Sanger sequencing. FBXW7R505C mutation was carried out by site-directed mutagenesis using pMRX-GFP-FBXW7 as a template and confirmed by Sanger sequencing. All transfections were performed using Lipofectamine 3000 reagent (Thermo Fisher Scientific), and selections were done in the presence of blasticidin hydrochloride (2 μg/ ml; HiMedia).

siRNA-directed gene knockdown

For RNA interference–mediated knockdown of specific genes, the following siRNAs were used: siFBW7 (Dharmacon ON-TARGET SMARTpool L-004246-00-0005), siCullin1 (Qiagen, 1027423), siSKP1 (Qiagen, SI00301819), and siGSK3β (Dharmacon ONTARGET SMARTpool L-003010-00-0005). Briefly, A549 cells grown in a 24-well plate were transiently transfected with gene-specific siRNAs or scramble (siControl) (150 pmol) using Lipofectamine 3000 as per the manufacturer’s instructions. Thirty-six hours after transfection, cells were processed for Western blotting and immunofluorescence or penicillin-gentamycin protection assays.

Structure prediction and modeling

All structures were predicted by AlphaFold (Protein Homology/analogy Recognition Engine V 2.0) (57) and visualized using PyMOL (The PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC). Structures were color-coded in PyMol based on IUPred (54) scores ranging from ordered (blue) to disordered (red) through white.

Western blotting

SPN cultures grown to 0.4 OD600nm were lysed by sonication, and crude extracts were collected following centrifugation (15,000 rpm, 30 min, 4°C). For A549s, monolayers were washed several times with PBS and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer [50 mM tris-Cl (pH 7.89), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 1% SDS] containing protease inhibitor cocktail (Promega), sodium fluoride (10 mM), and EDTA (5 mM). The cell suspension was briefly sonicated and centrifuged to collect cell lysates. Proteins present in bacterial or A549 cell lysates (10 or 20 μg) were separated on 12% SDS-PAGE gels and transferred to an activated polyvinylidene difluoride membrane. Following blocking in 5% skimmed milk, the membranes were probed with appropriate primary and HRP-tagged secondary antibodies. The blots were lastly developed using an enhanced chemiluminescence substrate (Bio-Rad).
Penicillin-gentamicin protection assay

SPN strains grown until OD600nm 0.4 in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) were pelleted, resuspended in PBS (pH 7.4), and diluted in assay medium for infection of A549 monolayers with a multiplicity of infection (MOI) of 10. Following 1 hour of infection, the monolayers were washed with DMEM and incubated with an assay medium containing penicillin (10 μg/ml) and gentamicin (400 μg/ml) for 2 hours to kill extracellular SPN. Cells were then lysed with 0.025% Triton X-100, and the lysate was plated on Brain Heart Infusion agar plates to enumerate viable SPN. Percentage invasion was calculated as [colonyforming units (CFU) in the lysate/CFU used for infection] × 100. To assess the intracellular survival, at 9 hours after infection (from the beginning of penicillin-gentamycin treatment), cell lysates were prepared as mentioned above, and spread plated and surviving bacteria were enumerated. Survival efficiency (%) was represented as a fold change in percent survival relative to control at indicated time point (normalized to 0 hours).

Immunofluorescence

For immunofluorescence assay, A549 or HeLa cells were grown on glass coverslips and infected with SPN or STm strains at MOI ~25 for 1 hour followed by antibiotic treatment for 2 hours. At desired time points after infection (9 hours for SPN infection in A549s and 3 hours for infection with STm in HeLa), cells were washed with DMEM and fixed with ice-chilled methanol at −20°C for 10 min. Further, the coverslips were blocked with 3% bovine serum albumin (BSA) in PBS for 2 hours at room temperature (RT). Cells were then treated with an appropriate primary antibody in 1% BSA in PBS overnight at 4°C, washed with PBS, and incubated with a suitable secondary antibody in 1% BSA in PBS for 1 hour at RT. Last, coverslips were washed with PBS and mounted on glassslides along with VECTASHIELD with or without 4′,6-diamidino-2-phenylindole (Vector Laboratories) for visualization using a laser scanning confocal microscope (LSM 780, Carl Zeiss) under 40× or 63× oil objectives. The images were acquired after optical sectioning and then processed using ZEN lite software (version 5.0). Superresolution microscopy was performed similarly using Elyra 7 (Carl Zeiss) in SIM mode. For colocalization analysis, bacteria were scored by visual counting of n > 100 bacteria per replicate.

Transmission electron microscopy

Following infection with SPN and STm, cells were washed with 0.1 M sodium cacodylate buffer, fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in 0.1 M sodium cacodylate buffer (Sigma-Aldrich) for 3 hours at 4°C. After fixation, cells were collected through a cell scraper and pelleted at 2000 rpm for 10 min. Cells were then washed with 0.1 M sodium cacodylate buffer and postfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 20 min at 4°C. Following subsequent washes with cacodylate buffer and distilled water, cells were then dehydrated in increasing concentrations of ethanol (50, 70, 95, and 100%) and propylene oxide for 30 min and lastly embedded in epoxy resin (Electron Microscopy Sciences, 14300) by polymerization at 75°C for 45 min. Subsequently, the capsules were transferred to a 95°C oven for 45 min. Ultrathin sections (70 nm) were cut using a diamond knife on a Leica EM UC7 Ultramicrotome and stained with 2% uranyl acetate and 1% lead citrate and viewed using a transmission electron microscope (Talos L120 C, Thermo Fisher Scientific) at 120 KV.

Ex vivo ubiquitination

A549-expressing BgaA-T and its variants were treated with MG132 (5 μM), and protein expressions were induced with doxycycline (100 μg/ml) for 24 hours. Cells were then lysed in RIPA buffer, and samples were electrophoresed by SDS-PAGE (8%). Confirmation of protein expression and ubiquitination of BgaA-T and its variants were proved by Western blot following probing with anti-BgaA and anti–K48-Ub antibodies, respectively.

In vitro ubiquitination

For in vitro ubiquitination assays, recombinant proteins were purified as described earlier (58). The SCFFBW7 E3 ligase complex was incubated with recombinant 0.1 mM E1 (UBE1; Boston Biochem), 0.25 mM E2 (cdc34; Boston Biochem), and ubiquitin (2.5 μg/ml; Boston Biochem) in the presence of purified BgaA-T and its variants. Ubiquitylation reactions were performed in assay buffer [50 mM tris (pH 8), 5 mM MgCl2, 5 mM adenosine triphosphate (ATP), 1 mM β-mercaptoethanol, and 0.1% Tween 20] for 2 hours at 25°C. The reactions were stopped with 5× Laemmli buffer, resolved on SDS-PAGE gels, and analyzed by immunoblotting using an anti-BgaA antibody.

In vitro kinase assay

To perform in vitro kinase assay, the GSK3β kinase enzyme system (Promega) was used as per the manufacturer’s protocol with the following modifications. Briefly, 3 μg of recombinant BgaA-T was added to 0.5 μg of active GSK3β with 400 μM ATP in reaction buffer composed of 40 mM tris, (pH 7.5), 20 mM MgCl2, BSA (0.1 mg/ml), and 50 μM dithiothreitol. The reaction was incubated for 2 hours at 30°C and stopped by adding 1× Laemmli buffer. The samples were then boiled and electrophoresed for Western blotting as mentioned previously. Phosphorylated BgaA-T was detected with an anti-phosphothreonine antibody.

In vivo model

All animal experiments were performed at the University of Liverpool in strict accordance with U.K. Home Office guidelines, under project license PP2072053, following approval from local animal welfare and ethics committees. For infection studies, 7- to 8-week female CD1 mice were purchased from Charles River Laboratories, United Kingdom, and allowed to acclimatize for 7 days before use. Briefly, mice were placed into a restraint tube, and S. pneumoniae was administered by intravenous injection into the tail vein (1 × 106 CFU in 100 μl of PBS). Mice were periodically scored for clinical signs of disease and culled when they showed signs of advanced pneumococcal disease or else at predetermined times after infection. Severity endpoints were defined as one or more of the following: substantially elevated or reduced respiratory rate, substantial reduction in natural behavior or moderate reduction in provoked behavior, loss of >20% starting body weight, and pronounced nasal or ocular discharge. Blood samples were obtained by cardiac puncture under terminal anesthesia, and spleens were excised postmortem for bacterial enumeration. Tissue samples were processed with a hand-held tissue homogenizer, and homogenates were serially diluted in PBS before spotting onto blood agar plates. Plates were incubated overnight at 37°C, 5% CO2, and bacterial colony numbers were assessed the following day.

Statistical analysis

GraphPad Prism version 5 was used for statistical analysis. Statistical tests undertaken for individual experiments are mentioned in the respective figure legends. P < 0.05 was considered to be statistically significant. Data were tested for normality and to define the variance of each group tested. All multiparameter analyses included corrections for multiple comparisons, and data are presented as means ± SD unless otherwise stated.

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