NagZ modulates the virulence of E. cloacae by acting through the gene of unknown function, ECL_03795

Figures & data

Table 1. Isolates and clinical data.

strains	patients’ information			sequence type	sample	infection symptoms before treatment	using antibiotics	after using antibiotics
	age	gender	diagnose					same pathogen detected again	symptoms of infection
WT1	72	male	sepsis	ST 696	blood	yes	yes	no	improved
WT2	68	female	cystitis	ST 422	urine	yes	yes	no	improved
WT3	53	male	traumatic infection	ST 414	wound secretion	yes	yes	no	improved

Table 2. Primers information.

Primers	Primer sequences		Product size(bp)
ECL_03795-qPCR Forward	5’GGGGTTATTGCCGATCTGTC 3’	136
ECL_03795-qPCR Reverse	5’GTTGTTGTCGAGCGCCTTTT 3’
ECL_02048-qPCR Forward	5’CCTGAACTGGCGAGAATGGC 3’	143
ECL_02048-qPCR Reverse	5’CCTGAACTGGCGAGAATGGC 3’
nagZ-CDS Forward	5’ATGTTGGATGTCGAAGGGTTTG 3’	1014
nagZ-CDS Reverse	5’TTAAAGGGCTGCTTTATGTGCC 3’
ECL_03795-CDS Forward	5’ATGAACACTATCGCTTTCCTG 3’	2241
ECL_03795-CDS Reverse	5’TCAGGCGTCCGCTACGGACG 3’
16S-qPCR Forward	5’TCCTACGGGAGGCAGCAGT 3’	467
16S-qPCR Reverse	5’GGACTACCAGGGTATCTAATCCTGTT 3’

qPCR: Real-time fluorescence quantitative polymerase chain reaction, CDS: coding DNA sequence.

Figure 1. Depletion of NagZ attenuates the inflammatory response of murine macrophages RAW264.7 to E. cloacae. (a) Variations in IL-6 expression at different time points following E. cloacae infection in murine macrophages RAW264.7. (b) Changes in TNF-α expression at different time points after E. cloacae infection in murine macrophages RAW264.7. * (p < 0.05) indicates a statistically significant, **(p < 0.01) denotes a high level of statistical significance and ***(p < 0.001) signifies extremely high statistical difference (p < 0.001). WT: wild type E. cloacae, ΔnagZ: nagZ knockout E. cloacae.

Figure 2. NagZ knockout diminishes E. cloacae virulence. (a, b) the survival rates of Galleria mellonella larvae (a) and mice (b) infected with nagZ-knockout E. cloacae are significantly improved compared to those infected with wild-type E. cloacae. (c, d) the expressions of IL-6 and TNF-α are downregulated in the serum of mice infected by nagZ-knockout E. cloacae, in contrast to wild-type E. cloacae. *p < 0.05 and ** p < 0.01 indicate statistically significant and statistically highly significant, respectively.

Figure 3. NagZ complementation partially rescues E. cloacae virulence in nagZ knockout models. (a,b) NagZ complementation decreases the survival rate of Galleria mellonella larvae (A) and mice (B). (c,d) the expression levels of IL-6 (C) and TNF-α (D) are upregulated in the serum of mice infected by E. cloacae complemented with NagZ. ΔnagZ+NagZ: ΔnagZ strains with NagZ complementation. * (p < 0.05) indicates a statistically significant, and **(p < 0.01) denotes a high level of statistical significance. NS: none statistical significance.

Figure 4. Transcriptome sequencing reveals NagZ’s involvement in nucleotide metabolism, specifically purine nucleotide metabolism. (a) NagZ knockout upregulates 24 genes and downregulates 86 genes in E. cloacae. (b) Gene ontology (GO) enrichment analysis assesses the impact of nagZ knockout on metabolic pathways in E. cloacae. (c) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis examines the effect of nagZ knockout on metabolic pathways in E. cloacae.

Figure 5. NagZ’s role in E. cloacae biofilm synthesis. (a) nagZ knockout enhances the synthesis of the second messenger c-di-GMP. (b) nagZ knockout increases E. cloacae biofilm synthesis, as observed through crystal violet staining. (c) Quantitative analysis of NagZ’s role in biofilm synthesis conducted via crystal violet staining experiments. (d) Detection of NagZ’s impact on E. cloacae adhesion to organic material (HEK-293T cells). * (p < 0.05) indicates a statistically significant, **(p < 0.01) denotes a high level of statistical significance and ***(p < 0.001) signifies extremely high statistical difference (p < 0.001).

Figure 6. Illustrating NagZ’s role in the expression levels of phosphodiesterases EC_02048 and EC_03795. (a) RT-qPCR results indicate that NagZ does not significantly affect the expression of EC_02048. (b) After NagZ knockout, there is a substantial downregulation in the expression level of phosphodiesterases EC_03795. (c) The expression level of phosphodiesterases EC_03795 is rescued upon complementation of NagZ in nagZ-knockout E. cloacae. e2048: ECL_03795 gene, e3795: ECL_03795 gene. * (p < 0.05) indicates a statistically significant, and **(p < 0.01) denotes a high level of statistical significance and ***(p < 0.001) signifies extremely high statistical difference (p < 0.001).

Figure 7. Examining the roles of NagZ and EC_03795 in E. cloacae virulence. (a) The survival rate of Galleria mellonella larvae assay assesses the impact of NagZ and EC_03795 on E. cloacae’s virulence. (b) Illustrates the functions of NagZ and EC_03795 in the expression of the second messenger c-di-GMP in E. cloacae. (c, d) evaluating the roles of NagZ and EC_03795 in the expression of the inflammatory factors IL-6 (C) and TNF-α (D) in a mouse infection model. ΔnagZ+E3795: ΔnagZ strain complemented with ECL_03795, Δe3795: WT1 strain with knockout of the ECL_03795 gene, Δe3795+E3795: Δe3795 strain complemented with ECL_03795. * (p < 0.05) indicates a statistically significant, **(p < 0.01) denotes a high level of statistical significance and ***(p < 0.001) signifies extremely high statistical difference (p < 0.001).

Data availability statement

The data that support the findings of this study are openly available in the figshare repository at https://doi.org/10.6084/m9.figshare.25898071.v1. The raw data of Transcriptome Sequencing data have been deposited in the GenBank repository under BioProject ID PRJNA1113565 (http://www.ncbi.nlm.nih.gov/bioproject/1113565)