Introduction

Cronobacter sakazakii (C. sakazakii), previously known as Enterobacter sakazakii, is a Gram-negative bacteria that can cause meningitis, septicaemia, and necrotizing enterocolitis in individuals of all ages1,2. Although infections in adults are usually less severe, neonates, especially those with low birth weights, are at greater risk3,4. There are reports of C. sakazakii infection in neonatal intensive care units worldwide, with mortality rates reported to be approximately 27%, as revised by Friedemann5. Survivors of meningitis may experience severe neurological sequelae such as hydrocephalus, tetraplegia, and developmental delay6,7. Infants who are immunocompromised, premature, have low birth weights, or younger than 28 days old are more vulnerable to infection than older infants are7,8,9,10.

C. sakazakii is frequently found in water, soil, and various food sources, such as plant materials11. The bacterium has developed resistance to desiccation and produces protective compounds such as extracellular polysaccharides and yellow carotenoids12,13. Neonatal infections caused by C. sakazakii are linked primarily to contaminated formula, likely because formula feeding is a common practice among newborns and other routes of exposure are limited14,15,16,17. However, infections have also been reported due to C. sakazakii contamination of breast pumps, emphasizing the potential for extrinsic contamination even in non-formula-fed infants. Cases include a premature infant in Pennsylvania infected through extrinsically contaminated expressed breast milk18, a similar infection in Australia19, and meningitis in a full-term neonate exclusively fed breast milk in Indiana20. Notably, this organism’s resistance to desiccation is crucial in its ability to survive and persist in formula and within infant formula manufacturing facilities.

The mechanisms underlying the pathogenicity of C. sakazakii are complex and not fully understood due to the diversity of virulence factors involved21. Adherence, invasion, and biofilm formation are extensively studied processes that contribute to the understanding of bacterial pathogenesis22,23. Studies have revealed the importance of the outer membrane proteins A and X (OmpA and OmpX) in the adherence and invasion of C. sakazakii in human intestinal and brain microvascular endothelial cells24. The RNA molecular chaperone Hfq also plays a crucial role in the biogenesis of outer membrane proteins and virulence25,26,27. Moreover, Cronobacter plasminogen activator (Cpa), an outer membrane protease, contributes to resistance to bactericidal serum and promotes invasion28. We previously reported that NlpD, an outer membrane lipoprotein of C. sakazakii, plays a significant role in the acid tolerance and pathogenicity of the bacterium29. However, little is known about the effect of NlpD on environmental tolerance.

In this study, C. sakazakii ATCC BAA-894, a well-characterized clinical isolate, was selected as the model strain due to its extensive genomic and proteomic annotations available in Uniprot30,31. This comprehensive proteomic database facilitates precise protein identification and functional analysis, ensuring the reliability of our findings. Moreover, this strain has been widely used in studies investigating stress tolerance, virulence, and antibiotic resistance, providing a reliable baseline for exploring the role of NlpD in desiccation resistance and biofilm formation13,32,33,34. Insights into biofilm formation and cellular adherence may further contribute to the development of novel antibiotics, as these processes play a critical role in bacterial persistence and infection. While the strain originates from a clinical setting, understanding its environmental adaptation mechanisms can also shed light on the broader ecological fitness of C. sakazakii, including its survival in food production environments where desiccation stress is common. Here, we examined the impact of nlpD deletion on desiccation tolerance, adherence, and motility, as well as compared the proteomic profiles of C. sakazakii ATCC BAA-894 and nlpD mutants.

Materials and methods

Bacterial strains, plasmids, and growth conditions

The various strains, plasmids, and primers used in this study are detailed in Table 1. The bacterial strains were stored in LB broth supplemented with 15% glycerol (final concentration) at -80 °C and revived before the experiments. Antibiotics were added as necessary to reach final concentrations of ampicillin (100 µg/mL) (INALCO, Inalco Pharmaceuticals, San Luis Obispo, CA) and kanamycin (50 µg/mL) (INALCO, Inalco Pharmaceuticals, San Luis Obispo, CA). Plasmid DNA and the pCVD442 suicide vector were prepared from E. coli DH5α and E. coli S17 lambda pir, respectively. In-frame deletion mutants were generated via the pCVD442 suicide vector method35. The low-copy vector pACYC184 was used for complementation36. C. sakazakii transformation and selection were performed via an established method32,37. All the plasmids were verified via DNA sequencing, and the primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Table 1 Bacterial strains, plasmids and primers used in this study.
Full size table

Desiccation resistance assays

Desiccation tolerance analysis was performed as previously reported36,38. Briefly, C. sakazakii and its mutant strains were cultured overnight in LB broth at 37 °C with shaking at 200 rpm until reaching the logarithmic growth phase. The resulting bacterial suspensions were subsequently transferred to 96-well plates and sealed with sealing film. The plates were placed in a sterilized desiccator containing 500 g of silica gel and incubated at 37 °C for 6 days. To record the initial cell counts, 10 µL of each bacterial suspension was serially diluted in PBS, with 10-fold dilutions performed, and inoculated onto PCA (Plate Count Agar) medium (Hopebio Technology Co., Ltd., Qingdao, China). To determine survival after desiccation, 100 µL of PBS was added to each dry sample and incubated at room temperature for 5 min. Bacterial cells were then suspended by pipetting and scraping from the surface of the 96-well plate, serially 10-fold-diluted in PBS, and inoculated onto PCA agar plates. After overnight incubation at 37 °C, bacterial colonies were counted. The percent desiccation inactivation was calculated as the proportion of bacteria that did not survive desiccation, expressed as a percentage, by subtracting the ratio of viable bacteria after desiccation to those before desiccation from 1, and multiplying by 100.

Biofilm formation

Crystal violet staining (CVS) was used to determine the biofilm-forming ability as previously described, with modifications38,39. C. sakazakii isolates were inoculated into 10 mL of LB broth and cultured at 37 °C with shaking at 200 rpm until the cell density reached 10⁷ CFU/mL. A 100 µL aliquot of the bacterial suspension was added to each well of a 96-well cell culture plate. The plates were incubated statically at 37 °C for 48 h. To immobilize the biofilm, 200 µL of 99% methanol was added for 15 min to remove the supernatant, and the plates were air-dried. Subsequently, 200 µL of 0.1% crystal violet (CV) solution was added. The excess CV was removed after 30 min of incubation, and the plates were washed with 0.9% NaCl. Finally, 200 µL of 95% ethanol was added to release the bound CV. The absorbance was measured at 570 nm via a microplate reader.

Motility assay

The motility assay was performed as previously described40,41. To assess motility, WT and mutant strains were grown to log phase in LB liquid media at 37 °C with shaking at 200 rpm. A 5 µL aliquot of the bacterial culture was spotted onto the center of soft agar motility plates (LB containing 0.3% agar) and allowed to dry at room temperature. The plates were then incubated for 12–14 h at 30 °C. Finally, the colony size was observed, and the average migration diameter was calculated based on the results of three independent experiments.

Adherence assay

The adherence assay was performed via the plate counting method38,41. Briefly, WT, ΔnlpD, and cp.nlpD C. sakazakii cells were cultured to an OD600 of 0.6, and then collected and washed three times with RPMI-1640 medium. The bacteria were resuspended in RPMI-1640 medium. Meanwhile, HCT-8 cells were cultured in 6-well tissue culture plates. After reaching the desired growth phase, the bacterial inocula were added to the HCT-8 cells, and the co-culture was incubated at 37 °C for 45 min in the presence of 5% CO2. After infection, the cells were washed three times with PBS, followed by lysis with 1% Triton X-100 (Abcone Biotech Co., Ltd. Beijing, China). The resulting cell suspensions were serially diluted and plated on LB agar to quantify the attached bacteria.

Membrane permeability

The outer membrane permeability of the cells was determined as previously described42. The C. sakazakii ATCC BAA-894 (WT), ΔnlpD and cp.nlpD strains in the log phase of growth were centrifuged at 12,000 rpm for 1 min, washed three times with PBS, and resuspended to an OD600 of 0.5. The fluorescence intensities of the WT and mutant suspensions were measured immediately by adding N-phenylnaphthalen-1-amine at a final concentration of 40 mM, using a fluorescence spectrophotometer (JASCO, Japan). Fluorescence was measured with excitation at 340 nm and emission at 420 nm.

Cell surface hydrophobicity

The cell surface hydrophobicity (CSH) was determined according to a previous report40. Briefly, cells were grown to the mid-exponential growth phase, and the bacterial suspensions were adjusted to an OD600 of 1.0 (H0). Subsequently, 2 mL of each of the bacterial suspensions were mixed with 0.4 mL of xylene and incubated at room temperature for 2 h. Finally, the OD600 of the aqueous phase was measured as H. The bacterial surface hydrophobicity index (H) was calculated as follows: H=(H0-H)/H0 × 100.

Protein extraction

The cells in the bacterial suspension were pelleted by centrifugation at 12,000 × g for 2 min at 4 °C and washed twice with PBS to remove residual culture medium. A protease inhibitor cocktail (Beyond Biotechnology Co., Ltd., China) was added to the lysed cells to prevent protein degradation, and ultrasonic waves (200 W) were applied for 10 min in 3-second working and 3-second pause cycles to lyse the cells. The resulting protein extracts were obtained by centrifugation at 21,000 × g for 15 min at 4 °C, and the supernatant was subsequently purified through a 0.22 μm microporous filter to remove impurities. The purified proteins were stored at -80 °C for subsequent mass spectrometry analysis.

Protein digestion

The preparation of protein samples for LC‒MS/MS analysis was carried out following a previously described protocol with a few modifications43,44,45,46. To prepare protein samples for LC‒MS/MS analysis, 50 µg of protein was denatured with 8 M urea (Aladdin Biochemical Technology Co., Ltd., Shanghai, China) and 50 mM ammonium bicarbonate (Aladdin Biochemical Technology Co., Ltd., Shanghai, China). The mixture was then reduced with DTT (Aladdin Biochemical Technology Co., Ltd., Shanghai, China), alkylated with iodoacetamide (Aladdin Biochemical Technology Co., Ltd., Shanghai, China), and digested overnight with trypsin (Thermo Fisher Scientific, Waltham, MA, USA). The digestion was halted with 10% trifluoroacetic acid (Aladdin Biochemical Technology Co., Ltd., Shanghai, China), and the samples were desalted using a C18 SPE (Merck Millipore, Burlington, MA, USA) column before being vacuum-dried and submitted for MS analysis.

LC‒MS/MS analysis

A Q Exactive Plus mass spectrometer and EASY nanoliquid chromatography system were used to analyse the peptide samples according to a previously described protocol43,44,45,46. The EASY nLC 1200 instrument, equipped with a Thermo Scientific Acclaim Pepmap nanotrap column and a Thermo Scientific EASY-Spray column, was run with the mobile phases (A: 0.1% formic acid; B: 80% CH3CN/0.1% formic acid) at 300 nL/min. The following gradients were used for liquid chromatography separation: 0–8% B for 3 min, 8–28% B for 42 min, 28–38% B for 5 min, and 38–100% B for 10 min. Eluted peptides were electrosprayed directly into the mass spectrometer for MS and MS/MS analysis in data-dependent acquisition mode. After one full MS scan (m/z 350–1500) was acquired, the 10 ions with the highest intensity for MS/MS analyses were immediately selected. A duration of 24 s and an exclusion duration of 12 s were set for dynamic exclusion. The mass spectra were compared against the UniProt database, and MaxQuant software (version 2.1.0.0) was used for data processing. Cysteine and methionine were set to fixed carbamidomethylation and variable oxidation, respectively. The specificity of trypsin cleavage, with a maximum of two possible missed cleavages, has a precursor mass tolerance of 10 parts per million and a fragment mass tolerance of 0.02 Da. Protein and peptide identification was based on a maximum false discovery rate of 1.0%, and protein quantification was calculated based on the median number of unique peptides for each protein.

GO and KEGG enrichment analyses

Enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed via the DAVID online database (https://david.ncifcrf.gov). The analysis results were automatically calculated with previously described methods to obtain enrichment scores.

Statistical analysis

Statistical analysis was performed via GraphPad Prism 9 to evaluate group differences47. Each experiment was independently repeated three times to ensure reproducibility. The significance of differences was determined via ANOVA. P values less than 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (****) were considered statistically significant.

Results

Construction and growth characteristics of the NlpD mutant in C. sakazakii

To assess the role of the nlpD gene (Fig. 1A) in the environmental tolerance and pathogenesis of C. sakazakii BAA-894, the generation of a nlpD knockout mutant (Locus_tag ESA_00549; coded_by CP000783.1:522560.523528, 968 bp) was carried out via the pCVD442 suicide plasmid technique (Fig. 1B). The loss of the nlpD gene was confirmed by PCR using the flanking primers ΔnlpD-E and ΔnlpD-F (sequences provided in Table 1). The PCR product was subjected to Sanger sequencing, with ΔnlpD-E and ΔnlpD-F used as sequencing primers. Additionally, transcriptional analysis of the downstream gene revealed that the deletion of nlpD does not affect the mRNA levels of the adjacent ESA_00550 (data not shown). We then tested the effect of nlpD on bacterial growth. The growth curves revealed that the ΔnlpD mutant largely overlapped with the wild-type growth curve, indicating that both strains had similar growth rates (Fig. 1B). The complementation strain (cp.nlpD) demonstrated a slightly greater growth rate than did the WT before stationary phase growth, but the difference was not significant (Fig. 1C). These results indicate that the deletion of the nlpD gene does not affect bacterial growth, indicating that it is a nonlethal gene of bacteria and is not necessary for cell growth.

Fig. 1
Fig. 1
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Construction and growth characteristics of the nlpD mutant in C. sakazakii. (A) Schematic representation of the genomic location of nlpD (ESA_00549). The stop codon of nlpD is located 50 bases upstream of the start codon of the adjacent downstream gene ESA_00550. (B) Construction of the pCVD442 suicide plasmid for nlpD deletion in C. sakazakii. (C) Growth of WT, ΔnlpD and cp.nlpD in LB medium. WT: C. sakazakii ATCC BAA-894; ΔnlpD: nlpD knockout mutant strain; cp.nlpD: complementation strain. Each data point represents the average and standard deviation of three biological replicates.

NlpD attenuates desiccation resistance

The role of the nlpD gene in the desiccation resistance of C. sakazakii was investigated by drying the C. sakazakii WT, ΔnlpD and cp.nlpD strains at 37 °C for 6 days. As shown in Fig. 2, the inactivation rates were 67.34%, 98.42%, and 85.47% for the WT, ΔnlpD and cp.nlpD strains, respectively, after 6 days of drying. Compared with that of the WT strain, the inactivation rate for the ΔnlpD strain was higher (p < 0.01), indicating the positive role of the nlpD gene in C. sakazakii desiccation resistance.

Fig. 2
Fig. 2
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The desiccation inactivation rates of the WT, ΔnlpD and cp.nlpD strains. WT: C. sakazakii ATCC BAA-894; ΔnlpD: mutant strain; cp.nlpD: Complementation strain. **p < 0.01. The data represent the results of three biological replicates.

NlpD affects biofilm formation

The ability to form biofilms is an important feature that enhances bacterial resistance to environmental stresses. Studies have suggested that NlpD is a lipoprotein that is involved in biofilm formation29. Therefore, to investigate the effect of the C. sakazakii nlpD gene on biofilm formation ability, we performed a crystal violet staining assay. The results revealed that the biofilm formation ability of the nlpD mutant was significantly lower than that of the WT strain, whereas the biofilm formation ability of the complemented strains was slightly lower than that of the WT strain (Fig. 3). These results indicate that the nlpD gene plays an important role in the formation of biofilms. In addition, the expression level of nlpD in cp.nlpD did not reach the WT level, possibly due to the use of the backbone pACYC184 in cp.nlpD, which is a low-copy-number plasmid. This phenomenon was also reported in previous papers29,42.

Fig. 3
Fig. 3
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Biofilm formation ability of C. sakazakii strains. The amount of biofilm formed was quantified by measuring the absorbance of solubilized crystal violet at 590 nm. WT: C. sakazakii ATCC BAA-894; ΔnlpD: mutant strain; cp.nlpD: Complementation strain. ***p < 0.001. The data represent the results of three biological replicates.

Effects of NlpD knockout on adherence and surface hydrophobicity

It is considered essential to have the ability of adherence to and invasion of tissue cells for the most pathogenic of bacteria. Our previous work revealed that NlpD is a novel virulence factor that is required for C. sakazakii survival in macrophages29. To investigate the impact of the nlpD gene on bacterial adherence to the HCT-8 cell line, we compared the adherence rate of the nlpD knockout strain with that of the wild-type strain. The results revealed that the nlpD knockout strain had a significantly lower adherence rate (Fig. 4A). Initial cell adherence, aggregation, and colony assembly are determined by the surface hydrophobicity of cell membranes, thus leading to biofilm development. Therefore, we compared the hydrophobicity of the surfaces of the WT and ΔnlpD mutant strains. The data indicated that the outer membrane hydrophobicity of the ΔnlpD mutant strain significantly decreased (Fig. 4B), suggesting that the nlpD gene plays a crucial role in in a cell’s surface hydrophobicity.

Fig. 4
Fig. 4
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Adherence rates (A) and surface hydrophobicity (B) of the WT, ΔnlpD and cp.nlpD strains. The data represent the results of three biological replicates. WT: C. sakazakii ATCC BAA-894; ΔnlpD: mutant strain; cp.nlpD: Complementation strain. ***p < 0.001 and ****p < 0.0001. The data represent the results of three biological replicates.

Evaluation of motility and membrane permeability

C. sakazakii can form a motility ring in semisolid media. For example, on 0.3% agar medium, WT, ΔnlpD and cp.nlpD strains can form different-sized swimming rings. Compared with the WT strain, the ΔnlpD mutant strain presented a significant difference in the motility radius, and the swimming mobility of the cp.nlpD strain was restored to some extent (Fig. 5A). As NlpD is a major outer membrane porin, the outer membrane permeability of C. sakazakii WT and the ΔnlpD mutant was assessed. As shown in Fig. 5B, the deletion of the nlpD gene did not affect membrane permeability.

Fig. 5
Fig. 5
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Motility assay (A) and membrane permeability (B) of the WT, ΔnlpD and cp.nlpD strains. WT: C. sakazakii ATCC BAA-894; ΔnlpD: mutant strain; cp.nlpD: Complementation strain. ***p < 0.001. The data represent the results of three biological replicates.

GO and KEGG pathway analysis

We systematically monitored the protein expression profiles of the C. sakazakii WT and ΔnlpD strains via label-free relative quantitative mass spectrometry. Through proteomic analysis, 1447 proteins were detected, and we found that nlpD knockdout significantly increased the abundance of 159 proteins while decreasing the abundance of 157 proteins (Fig. 6A, Table S1). The differentially expressed proteins were subjected to GO and KEGG pathway analyses. GO analysis revealed that the upregulated proteins were significantly enriched in molecular functions related to oxidoreductase activity, zinc ion binding, and NAD binding. In addition, the upregulated proteins were enriched in carbohydrate metabolic processes. The downregulated proteins were enriched in seven biological function-related clusters and three molecular function-related clusters (Fig. 6B). Both upregulated and downregulated proteins were found in the cellular component of the cytoplasm. KEGG pathway enrichment analysis revealed that the downregulated proteins were significantly enriched in eight pathways (p < 0.05), and the upregulated proteins were enriched in six pathways, including three identical pathways related to metabolic pathways, biosynthesis of antibiotics, and biosynthesis of secondary metabolites (Fig. 6C). Among the eight pathways downregulated, “metabolism” was the most common, including several subclasses: metabolic pathways, starch and sucrose metabolism, carbon metabolism, glyoxylate and dicarboxylate metabolism, and pyruvate metabolism. These findings provide valuable insights into the impact of nlpD knockdout on the proteomic landscape of C. sakazakii, suggesting significant alterations in metabolic and biosynthetic pathways.

Fig. 6
Fig. 6
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Proteomic analysis of the nlpD gene knockout strain. (A) Volcano plot showing the impact of nlpD knockout on protein abundance. The criteria for significant changes were defined as a fold change greater than 2 and a P value of less than 0.05. Significantly upregulated proteins are represented by red dots (UP), significantly downregulated proteins are represented by green dots (DW), and proteins without significant differences are represented by grey dots (Nodiff). The number of proteins in each category is labeled in the figure. (B) Gene Ontology (GO) analysis of the significantly upregulated proteins caused by nlpD knockout. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the significantly upregulated proteins caused by nlpD knockout in metabolic pathways62,63,64. The size of the shape represents the number of proteins classified in that category, whereas the colour represents the ratio of the number of proteins classified in that category to the total number of proteins in that category.

Discussion

NlpD is a highly conserved gene, and the presence of homologous genes in various bacteria suggests that NlpD has an important function in bacteria. The Yersinia pestis NlpD lipoprotein has been reported to be involved in the regulation of cellular morphogenesis, and disruption of the nlpD of Yersinia pestis affects iron acquisition and the activity of the arginine transport system48,49. Tsang et al. reported that in Escherichia coli, NlpD acts as a bridge linking cell wall remodelling and outer membrane invagination during cytokinesis50. In Neisseria meningitidis, the AmiC/NlpD pathway dominates peptidoglycan breakdown and affects cell separation, NOD1 agonist production, and infection51. However, previous studies have focused on the relationships between NlpD and cell envelope integrity and iron acquisition, and these studies have suggested that NlpD plays a vital role in cell membrane integrity and nutrient acquisition in bacteria. Only a few studies have suggested that NlpD may be related to bacterial virulence29. Therefore, investigating the specific role of NlpD in bacterial pathogenicity is intriguing.

Cronobacter sakazakii is an emerging pathogenic bacteria in the family Enterobacteriaceae that has attracted attention due to its multidrug resistance and high mortality pathogenicity15. C. sakazakii NlpD is a novel virulence factor. Our previous study revealed that NlpD plays an essential role in bacterial acid tolerance, leading to macrophage tolerance and increased bacterial virulence29. However, this study focused only on the role of NlpD in the mechanism of acid tolerance, and little is still known about how NlpD affects other proteins involved in the pathogenic process of bacteria.

Here, we examined the impact of nlpD deletion on desiccation resistance, biofilm formation, adherence, cell surface hydrophobicity, and motility and compared the proteomic profiles of C. sakazakii ATCC BAA-894 and its nlpD mutants. In the present study, the ability of the nlpD-deleted strain to resist desiccation and form biofilms decreased significantly. Previous studies have reported that biofilm formation is an important means of protecting bacteria from long-term survival in dry environments, indicating that the nlpD gene may be involved in biofilm formation38,42. As a defence barrier and an important adherence base for biofilm cells, biofilms have been shown in many studies to play crucial roles in the adherence and invasion of human epithelial cells by pathogenic bacteria52,53,54,55. Considering that the C. sakazakii nlpD mutant presented a decreased biofilm formation phenotype, we tested the adherence and invasion of the WT, ΔnlpD and cp.nlpD strains by using HCT-8 cells to determine the effect of nlpD on cell adherence. Compared with that of the parent strain, the ability of the mutant strain to adhere to HCT-8 cells was dramatically decreased, indicating that nlpD may play a positive role in adhering to tissue cells. However, a previous study reported no effect of NlpD on adherence29, which may be due to differences in the cell lines used. C. sakazakii contains several genes that promote adherence, such as ESA_00986, which plays a positive role in adherence, invasion, and virulence37, and envZ/ompR, which is crucial for adherence and invasion32. Our study identified NlpD as a lipoprotein that may directly influence cell membrane interactions. For example, in eukaryotic cells, lipoprotein lipase enhances human monocyte adherence to aortic endothelial cells56 and carbamylated low-density lipoprotein induces monocyte adherence to endothelial cells57. However, the effects of chemical modification of bacterial lipoproteins on adherence require further investigation. The attachment of bacteria to a substance is the initial step in biofilm formation, and cell surface hydrophobicity greatly facilitates biofilm adherence. After the knockout of nlpD, the cell surface hydrophobicity was noticeably reduced, further suggesting a relationship between nlpD and biofilm formation.

In our study, the functions of the nlpD gene were investigated via the gene knockout technique in C. sakazakii. We found that this gene plays a positive regulatory role in desiccation resistance and biofilm formation. Furthermore, the results also revealed a significant role for nlpD in adherence and cell surface hydrophobicity. We identified a relationship between NlpD and the desiccation tolerance and adherence ability of C. sakazakii. C. sakazakii is a bacterium capable of causing gastrointestinal infections, and successful infestation requires the ability to resist desiccation in formula as well as its ability to successfully colonize the intestinal tract. This discovery reveals that NlpD may be a good target for antibiotic development because by interfering with the function of the outer membrane protein NlpD, it is possible to affect desiccation resistance, biofilm formation, adherence capacity, and acid tolerance, which are important processes in the pathogenic pathway of bacteria.

Our study used label-free relative quantitative mass spectrometry to systematically monitor the protein expression profiles of the C. sakazakii WT and ΔnlpD strains. Through proteomic analysis, we identified a total of 1447 proteins, and our findings revealed significant alterations in protein abundance upon nlpD knockdout. The upregulation of proteins associated with oxidoreductase activity, zinc ion binding, and NAD binding suggests a significant impact on the redox balance of the bacteria, because zinc can influence oxidoreductase activity and reactive oxygen species58. These findings imply a shift in energy metabolism or adaptation to stress conditions in response to nlpD knockdout. Additionally, the enrichment of upregulated proteins in carbohydrate metabolism processes suggests a potential alteration in the utilization of carbon sources, which could be vital for bacterial growth and survival. The diversity of downregulated proteins distributed across various biological function-related clusters highlights the multifaceted role of NlpD in regulating C. sakazakii cellular functions. This downregulation spans several metabolic pathways, indicating a broader impact on the metabolic activities of the bacteria. “Metabolism” emerged as a predominant category among the downregulated pathways, highlighting the central role played by NlpD in governing the metabolic machinery of C. sakazakii. Similar to our study, a previous study revealed that deletion of the flagellar gene fliF led to reduced pathogenicity and alterations in several metabolic pathways59, including “nucleotide transport and metabolism,” “carbohydrate transport and metabolism,” “coenzyme transport and metabolism,” and “lipid transport and metabolism.” In comparison, while the deletion of C. sakazakii nlpD also caused metabolic changes, these changes were related primarily to carbohydrates such as starch, sucrose, glyoxylate, dicarboxylate, and pyruvate, possibly highlighting the specific role of NlpD.

Although proteomics revealed extensive links between NlpD and metabolism, the relationships between the numerous up- and downregulated proteins caused by nlpD deletion and the phenotypic changes in desiccation resistance, biofilm formation, and cellular adherence remain to be fully elucidated. This could be achieved by analysing the connections between multiple gene knockouts and comparative proteomics, allowing for the identification of proteins influencing specific phenotypes. This approach has successfully revealed the role of C. sakazakii pyridoxal kinase PdxY, which is mediated by TreR and pESA3, in maintaining vitamin B6 (PLP) levels and virulence33, as well as the importance of maltodextrin-binding protein in C. sakazakii survival under desiccation stress36. Notably, the deletion of nlpD led to a decrease in the abundance of LprI, which, in Mycobacterium tuberculosis, displays antilysosomal activity and is considered a virulence factor60. We speculate that LprI may also act as a lysozyme inhibitor in C. sakazakii and that its downregulation could play a key role in the reduced virulence observed following nlpD deletion. Additionally, the protective role of trehalose in C. sakazakii under desiccation has been well documented in previous studies13,61. In this study, the deletion of nlpD led to a decrease in the abundance of trehalose 6-phosphate phosphatase, which may be a key mechanism by which NlpD affects C. sakazakii.

This research advances our understanding of the molecular mechanisms underlying the regulatory role of nlpD in C. sakazakii, particularly in its environmental tolerance and survival under adverse conditions such as desiccation. The insights gained from this study have practical implications for the food industry, as nlpD contributes to key phenotypes like biofilm formation and desiccation resistance, which are critical for the persistence of C. sakazakii in powdered infant formula production environments. Targeting nlpD-regulated pathways could inform strategies to mitigate contamination risks and improve sanitation protocols in industrial settings. Although this study primarily focuses on environmental tolerance rather than pathogenicity, further exploration of the identified proteins and pathways may uncover novel targets for interventions aimed at reducing C. sakazakii survival and virulence in food-related contexts.

Conclusion

This study illuminates the critical role of the NlpD protein in Cronobacter sakazakii, particularly in enhancing environmental tolerance. Through comprehensive evaluations of various bacterial behaviours and characteristics, including desiccation resistance, biofilm formation, and cellular adherence, NlpD clearly plays a pivotal role in these processes. The comparison between the nlpD mutant and wild-type strains highlighted significant deficiencies in the environmental adaptability of the mutant strain, underscoring the importance of NlpD in maintaining robust bacterial fitness. Proteomic analyses further revealed widespread alterations in protein expression pathways upon nlpD knockdout, emphasizing the molecular mechanisms through which NlpD influences bacterial physiology. These findings not only enhance our understanding of the pathogenicity of C. sakazakii but also suggest that NlpD is a promising target for future antimicrobial strategies aimed at combating this pathogen.