Impact of LuxS on virulence and pathogenicity in Klebsiella pneumoniae exhibiting varied mucoid phenotypes

ABSTRACT

How the LuxS/AI-2 quorum sensing (QS) system influences the pathogenicity of K. pneumoniae is complicated by the heterogeneity of the bacterial mucoid phenotypes. This study aims to explore the LuxS-mediated regulation of the pathogenicity of K. pneumoniae with diverse mucoid phenotypes, including hypermucoid, regular-mucoid, and nonmucoid. The wild-type, luxS knockout, and complemented strains of three K. pneumoniae clinical isolates with distinct mucoid phenotypes were constructed. The results revealed the downregulation of virulence genes of regular-mucoid, and nonmucoid but not hypermucoid strains. The deletion of luxS reduced the pathogenicity of the regular-mucoid, and nonmucoid strains in mice; while in hypermucoid strain, luxS knockout reduced virulence in late growth but enhanced virulence in the early growth phase. Furthermore, the absence of luxS led the regular-mucoid and nonmucoid strains to be more sensitive to the host cell defense, and less biofilm-productive than the wild-type at both the low and high-density growth state. Nevertheless, luxS knockout enhanced the resistances to adhesion and phagocytosis by macrophage as well as serum-killing, of hypermucoid K. pneumoniae at its early low-density growth state, while it was opposite to those in its late high-density growth phase. Collectively, our results suggested that LuxS plays a crucial role in the pathogenicity of K. pneumoniae, and it is highly relevant to the mucoid phenotypes and growth phases of the strains. LuxS probably depresses the capsule in the early low-density phase and promotes the capsule, biofilm, and pathogenicity during the late high-density phase, but inhibits lipopolysaccharide throughout the growth phase, in K. pneumoniae.

IMPORTANCE

Characterizing the regulation of physiological functions by the LuxS/AI-2 quorum sensing (QS) system in Klebsiella pneumoniae strains will improve our understanding of this important pathogen. The genetic heterogeneity of K. pneumoniae isolates complicates our understanding of its pathogenicity, and the association of LuxS with bacterial pathogenicity has remained poorly addressed in K. pneumoniae. Our results demonstrated strain and growth phase-dependent variation in the contributions of LuxS to the virulence and pathogenicity of K. pneumoniae. Our findings provide new insights into the important contribution of the LuxS/AI-2 QS system to the networks that regulate the pathogenicity of K. pneumoniae. Our study will facilitate our understanding of the regulatory mechanisms of LuxS/AI-2 QS on the pathogenicity of K. pneumoniae under the background of their genetic heterogeneity and help develop new strategies for diminished bacterial virulence within the clinical K. pneumoniae population.

INTRODUCTION

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative pathogen that can cause a variety of diseases including urinary tract infections, sepsis, liver abscesses, and pneumonia. The genetic background and mucoid phenotype of K. pneumoniae differ (1). The capacity to synthesize extracapsular polysaccharide (EPS) results in a variety of K. pneumoniae mucoid phenotypes (2, 3). Encapsulated strains of K. pneumoniae are fairly prevalent. Overproduction of capsule (CPS) contributes to hypermucoviscosity (HMV), one of the phenotypes associated with hypervirulent strains (4), whereas unencapsulated strains generally present as nonmucoid phenotypes. The high diversity of surface polysaccharides complicates our understanding of the varied physiological activities of K. pneumoniae of distinct mucoid phenotypes.

Autoinducer-2 (AI-2) is a quorum sensing (QS) signaling molecule, which is synthesized by LuxS (5). LuxS/AI-2 QS regulates bacterial characteristics such as surface attachment and/or biofilm development, motility, and virulence (6). The potential role of LuxS/AI-2 QS in virulence has been studied in several pathogens, including Serratia, Vibrio, Neisseria, Salmonella, and Haemophilus, and has been linked to bacterial virulence and biofilm formation (7). However, it is unclear how this gene is linked to a wide range of physiological functions in K. pneumoniae, especially for diverse mucoid phenotypes bacteria. More importantly, QS is known to sense cell density to regulate the biological function of bacteria (8–10). The exact regulatory mechanism of LuxS associated with the cell density in K. pneumoniae of diverse mucoid phenotypes remains unknown.

In this study, global gene regulation by LuxS/AI-2 in K. pneumoniae was investigated by comparing gene expression of wild-type and their isogenic ΔluxS mutant via transcriptome analysis in K. pneumoniae strains with different mucoid phenotypes. The effects of luxS mutation on bacterial virulence and pathogenicity were studied via phenotypic investigations in vivo and in vitro. Our results suggested that the contribution of LuxS to the virulence and pathogenicity of K. pneumoniae may be both strain and growth-state dependent.

RESULTS

K. pneumoniae FK1979, FK6768, and FK563 comprehensively represented three heterogeneous mucoid phenotypes of K. pneumoniae clinical isolates

Genetic diversity of three K. pneumoniae strains

The genetic characteristics of the three clinical K. pneumoniae strains are shown in Table 1.

TABLE 1.

The genetic characteristics of K. pneumoniae FK1979, FK6768, and FK563

Isolates	ST	K locus	Genetic information			Resistance genes	Virulence genes
			Number	Size	Replicon type
FK1979	ST86	K2	chromosome	5,322,984 bp	–	oqxB, oqxA, blaSHV-28, fosA	fyuA, ybtE, ybtT, ybtU, irp1, irp2, ybtA, ybtP, ybtQ, ybtX, ybtS, ompA, entA, entB, fepC, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ykgK/ecpR
			p1979-1a	227,097 bp	IncHI1B, repB	–	iutA, iucC, iucB, iucA, iroB, iroC, iroD, iroN
FK6768	ST11	KL64	Chromosome		–	blaSHV-182, aadA2, fosA6	fyuA, ybtE, ybtT, ybtU, irp1, irp2, ybtA, ybtP, ybtQ, ybtX, ybtS, ompA, entA, entB, fepC, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ykgK/ecpR
			p6768-1a	219,459 bp	IncHI1B, repB	–	iucA, iucB, iucC, iutA
			p6768-2a	77,979 bp	–	dfrA14, sul2, tet(A)	–
			p6768-3a	73,746 bp	IncFII, IncR	blaSHV-12, blaKPC-2	–
			p6768-4a	12,001 bp	ColRNAI	–	–
			p6768-5a	5,597 bp	–	–	–
FK563	ST37	KL14	Chromosome	5,347,560 bp	–	aph(3')-IIa, oqxB, oqxA, blaSHV-110, blaCTX-M-14, fosA6	fyuA, ybtE, ybtT, ybtU, irp1, irp2, ybtA, ybtP, ybtQ, ybtX, ybtS, ompA, entA, entB, fepC, yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ykgK/ecpR
			p563-1a	154,204 bp	IncFIB, IncFIA, IncFII, Col156	blaTEM-1B, aac (3)-IId, tet(A), aph (6)-Id, aph(3'')-Ib, sul2, mph(A), sul1, aadA5, dfrA17	senB
			p563-2a	94,216 bp	IncFII(K)	blaCTX-M-15, blaTEM-1B	–
			p563-3a	70,069 bp	IncFII	bla CTX-M-14	–
			p563-4a	4,245 bp	–	catA1, tet(C)	–

^a P represents plasmid.

Phenotypes of K. pneumoniae FK1979, FK6768, and FK563

Visual observations of colony morphology and string test showed that FK1979 was mucoid, moist, protruding, and sticky with a strong positive string test; FK6768 was smooth, cream-colored with a negative string test; FK563 strain appeared dry, translucent and had a comparably smaller colony size on blood agar with a negative string test (Fig. 1A). In addition to the visual observations, we performed the low-speed centrifugation assay to assess the mucoviscosity. FK1979 supernatants remained quite muddy throughout centrifugation, whereas FK6768 and FK563 were virtually clear. Significant differences in the supernatant absorbance [optical density at 600 nm (OD₆₀₀)] were observed, which was correlated with colony morphology (Fig. 1B). Due to the highly yielded CPS, hypervirulent hypermucoid strains resist serum killing (11). Direct quantification of the CPS production of strains indicated the highest CPS production in FK1979, producing significantly lower CPS in FK6768, while the CPS yield in FK563 was the lowest among the three strains (Fig. 1C). Many studies defined the hypervirulent traits through serum resistance assays (12). The changes in the number of survival bacteria in serum over time indicated that the two regular-mucoid and nonmucoid strains, FK6768 and FK563, were sensitive to serum killing, while the hypermucoid strain FK1979 exhibited resistance to serum (Fig. 1D). The 50% lethal dose (LD₅₀), 90% lethal dose (LD₉₀), and minimum lethal dose (MLD) against ICR mice at 7 days of FK6768 and FK563 were almost equal, which were approximately 1,000-fold higher than that of FK1979 (Fig. 1E; Table 2). Based on the characteristics of phenotype and genetics, FK6768, FK563, and FK1979 were considered to be low-virulent regular-mucoid, low-virulent nonmucoid, and hypervirulent hypermucoid phenotype, respectively. Overall, K. pneumoniae FK1979, FK6768, and FK563 represented three distinct phenotypes of K. pneumoniae clinical isolates. Analysis of the impact of luxS on K. pneumoniae strains of different genetic heterogeneity may help us better understand the comprehensive regulative effects of LuxS/AI-2 QS on K. pneumoniae pathogenicity.

Fig 1.

Mucoid and virulence phenotypes of the three clinical K. pneumoniae isolates. (A) Colony morphology and string tests of the strains cultured on blood agar plates. (B) Centrifugation analysis of the strains. NTUH-K2044 used as hypermucoviscosity control. Data were shown as mean ± SD. ns. not significant; ***, P < 0.0001 (Student’s t test compared to NTUH-K2044); ###, P < 0.0001 (Student’s t test compared to FK1979); …ns, not significant (Student’s t test compared to FK6768). (C) Quantification of the CPS production of strains. ns. not significant; ***, P < 0.0001 (Student’s t test compared to NTUH-K2044); ###, P < 0.0001 (Student’s t test compared to FK1979); …, P < 0.0001 (Student’s t test compared to FK6768). (D) Serum resistance analysis of the bacteria. The initial inoculation of approximately 10⁶ CFU/mL was 1:3 mixed with fresh serum and the CFU/mL was tested every 1 h for a total of 3 h. Repeated measures analysis of variance (ANOVA) was employed to assess the significance of differences. ns, not significant between FK6768 and FK563; ***, P < 0.001 compared to FK1979. (E) Dose-response curves for mice infection, the LD₅₀ and MLD were marked. Images were representatives of three independent experiments.

TABLE 2.

LD₅₀ and LD₉₀ values of K. pneumoniae FK1979, FK6768, and FK563 strains in mice

K. pneumoniae strains	Challenge dose (CFU)	Percent of dead mice (%)	Value of LD50 CFU	Value of LD90 CFU
FK1979	1.25 × 102	0 (0/5)	9.10 × 103	2.39 × 104
	1.06 × 103	20 (1/5)
	0.98 × 104	60 (3/5)
	1.23 × 105	100 (5/5)
	1.44 × 106	100 (5/5)
FK6768	1.15 × 105	0 (0/5)	2.06 × 107	7.14 × 107
	1.09 × 106	0 (0/5)
	0.97 × 107	20 (1/5)
	1.17 × 108	100 (5/5)
	1.06 × 109	100 (5/5)
FK563	1.14 × 105	0 (0/5)	3.43 × 107	8.86 × 107
	0.99 × 106	0 (0/5)
	1.17 × 107	0 (0/5)
	1.06 × 108	100 (5/5)
	1.13 × 109	100 (5/5)

The luxS gene is associated with the bacterial virulence and pathogenicity of K. pneumoniae

To explore the possible effects of LuxS on bacterial virulence and pathogenicity, we performed transcriptome sequencing analysis of the wild-type, △luxS strains in the log growth state and found that the expression of virulence and biofilm-related genes of △luxS mutations of FK6768 and FK563 were significantly lower than those of the wild-type strains. Of note, for FK1979, the expression of these genes in the log growth state had no significant difference (Tables 3 to 6). To affirm that LuxS affects bacterial virulence and pathogenicity based on the results of the transcriptome above, we subsequently investigated the impact of the luxS mutations on the in vivo virulence of the K. pneumoniae strains using mice infection models. First, we excluded that the effect of LuxS on bacterial pathogenicity was based on the effect of LuxS on bacterial growth. Growth curves (determined using OD₆₀₀) indicated that the mutants did not display a growth defect in vitro in Luria-Bertani (LB) medium (Fig. S1).

TABLE 3.

K. pneumoniae genes involved in virulence that are regulated by luxS at mid-log phase^b

		Fold change (ΔluxS mutant/WTa)
Genes	Putative identification of KEGG database	K. pneumoniae FK1979	K. pneumoniae FK6768	K. pneumoniae FK563
entH		–	0.16	0.66
entA	2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase	1.68	0.17	0.61
entB	Bifunctional isochorismate lyase/aryl carrier protein	1.04	0.27	0.44
entE	2,3-dihydroxybenzoate-AMP ligase	1.08	0.17	0.61
entC	Isochorismate synthase	1.22	0.20	0.56
fepB	Siderophores bacterioferritin (siderophore enterobactin)	–	0.39	0.37
entS	MFS transporter, ENTS family, enterobactin (siderophore) exporter	1.64	0.12	0.30
fepD	Siderophores bacterioferritin (siderophore enterobactin)	–	0.54	0.27
fepG	Siderophores bacterioferritin (siderophore enterobactin)	–	0.31	0.28
fepC	Siderophores bacterioferritin (siderophore enterobactin)	–	0.53	0.48
entF	Enterobactin synthetase component F	2.34	0.09	0.43
entD	Enterobactin synthetase component D	1.65	0.21	–
iucA	Aerobactin synthetases	1.05	0.33	–
iucB	Aerobactin synthetases	0.80	0.30	–
iucC	Aerobactin synthetases	1.14	0.26	–
iucD	Aerobactin synthetases	0.89	0.26	–
irp5	Yersiniabactin synthetases	1.37	0.47	0.71
irp4	Yersiniabactin synthetases	1.10	0.29	0.48
irp3	Yersiniabactin synthetases	1.05	0.13	0.53
irp1	Yersiniabactin synthetases	1.25	0.27	0.51
irp2	Yersiniabactin synthetases	1.10	0.37	0.64
irtA		0.92	0.22	0.70
ybtX	Yersiniabactin synthetases	1.21	0.29	0.47
intB		–	2.22	–
yeeO		–	0.76	–
ybdZ		–	0.20	–
fes		–	0.17	–
fepA.1	Siderophores bacterioferritin	–	0.42	–
iutA.1		–	1.11	–
fyuA		–	0.85	–
pchR		–	0.89	–
ybtS	Yersiniabactin synthetases	–	–	1.12
ybtP	Yersiniabactin synthetases	–	–	0.74
ybtQ	Yersiniabactin synthetases	–	–	0.69
irp9,ybtS	Yersiniabactin synthetases	1.18	–	–
mtfA		0.82	–	–
ompN		0.90	–	–

^a Normalized to 1.

^b Bold indicates that the difference is statistically significant.

TABLE 4.

K. pneumoniae genes involved in capsule that are regulated by luxS at mid-log phase^b

		Fold change (ΔluxS mutant/WTa)
Genes	Putative identification of KEGG database	K. pneumoniae FK1979	K. pneumoniae FK6768	K. pneumoniae FK563
galF	UTP--glucose-1-phosphate uridylyltransferase	0.97	1.78	–
cpsACP		–	2.54	–
wzi		1.13	1.44	–
wza		0.98	1.98	0.66
gtr3	Polysaccharide biosynthesis/export protein	0.96	2.34	0.50
gtr4	Glycosyltransferase	0.93	0.95	0.39
etk-wzc	Tyrosine-protein kinase Etk/Wzc	0.97	0.65	0.44
gtr5	Glycosyltransferase	1.35	1.21	0.41
gtr6	Glycosyltransferase	1.02	0.89	0.57
gtr7	Glycosyltransferase	1.32	1.16	0.47
wzxC		1.50	0.72	0.63
gtr8	Lipopolysaccharide exporter	1.40	1.14	0.43
gtr9	Glycosyltransferase	1.31	1.21	0.51
glf	Putative colanic acid biosysnthesis UDP-glucose lipid carrier transferase	–	–	0.58
OEW01_RS08160	6-phosphogluconate dehydrogenase	–	–	0.64
wcaJ	Mannose-1-phosphate guanylyltransferase	1.00	0.71	0.51
gnd	Phosphomannomutase	1.23	2.60	0.39
manC	UDPglucose 6-dehydrogenase	0.93	1.11	–
manB	UTP--glucose-1-phosphate uridylyltransferase	1.17	0.58	–
rfbB.1	Polysaccharide biosynthesis/export protein	–	1.38	–
rfbA	Polysaccharide biosynthesis/export protein	–	0.73	–
rfbD	Polysaccharide biosynthesis/export protein	–	0.48	–
rfbC	Polysaccharide biosynthesis/export protein	–	1.18	–
ugd	UDP-glucose dehydrogenase	1.28	1.53	–

^a Normalized to 1.

^b Bold indicates that the difference is statistically significant.

TABLE 5.

K. pneumoniae genes involved in biofilm formation that are regulated by luxS at mid-log phase^b

		Fold change (ΔluxS mutant/WTa)
Genes	Putative identification of KEGG database	K. pneumoniae FK1979	K. pneumoniae FK6768	K. pneumoniae FK563
fimH	Minor fimbrial subunit	0.84	0.32	0.53
fimG	Minor fimbrial subunit	1.11	0.23	–
fimF	Minor fimbrial subunit	0.86	0.18	1.60
fimD	Uter membrane usher protein	1.06	0.35	0.46
fimC	Fimbrial chaperone protein	0.71	1.24	1.21
fimI	Fimbrial protein	0.81	1.14	0.33
fimA	Major type 1 subunit fimbrin (pilin)	0.72	2.76	0.48
fimE	Type 1 fimbriae regulatory protein FimE	1.42	0.23	–
fimB	Type 1 fimbriae regulatory protein FimB	0.76	1.49	1.17
fimA	Major type 1 subunit fimbrin (pilin)	0.57	–	0.59
fimD, fimC, mrkC, htrE, cssD	Uter membrane usher protein	1.10	–	1.26
fimA	Major type 1 subunit fimbrin (pilin)	0.99	–	–
yhjH	c-di-GMP phosphodiesterase	0.66	–	0.81

^a Normalized to 1.

^b Bold indicates that the difference is statistically significant.

TABLE 6.

K. pneumoniae genes involved in LPS biosynthesis that are regulated by luxS at mid-log phase^b

		Fold change (ΔluxS mutant/WTa)
Genes	Putative identification of KEGG database	K. pneumoniae FK1979	K. pneumoniae FK6768	K. pneumoniae FK563
manC	Lipopolysaccharide biosynthesis protein	0.93	1.11	1.47
manB(rfbK1)	Lipopolysaccharide biosynthesis protein	1.17	0.58	3.57
rfbB.1	Lipopolysaccharide biosynthesis protein	–	1.38	3.53
rfbA	Lipopolysaccharide biosynthesis protein	–	0.73	2.02
rfbC	Lipopolysaccharide biosynthesis protein	–	1.18	3.04
ugd	Lipopolysaccharide biosynthesis protein	1.28	1.53	0.57
wcaG	Lipopolysaccharide biosynthesis protein	–	2.48	–
wzm	Lipopolysaccharide biosynthesis protein	–	2.28	–
rfbB	Lipopolysaccharide biosynthesis protein	0.99	2.06	0.65
wbbM	Lipopolysaccharide core biosynthesis glycosyltransferase	1.09	1.91	1.29
rfbD	Lipopolysaccharide biosynthesis protein	0.95	2.67	3.55
wbbN	Lipopolysaccharide core biosynthesis glycosyltransferase	0.98	1.98	1.71
wbbO	Lipopolysaccharide core biosynthesis glycosyltransferase	1.15	1.75	1.92

^a Normalized to 1.

^b Bold indicates that the difference is statistically significant.

LuxS contributes to the pathogenicity of low-virulent regular-mucoid K. pneumoniae FK6768, and low-virulent nonmucoid K. pneumoniae FK563 in the mouse infection model

LD₅₀ on mice model

To examine virulence in vivo, we infected ICR mice intraperitoneally (i.p.) with various doses of K. pneumoniae strains, including wild-type, ΔluxS, and C-luxS strains, to assess the impact of the luxS gene on virulence. The results showed that the mice in the high-dose group developed symptoms such as depression, rough fur, trembling, and collapse 6 h after injection, and most of the mice died within 3 days. For FK6768, the final LD₅₀ value of ΔluxS strain was 4.92 times as high as that required by the wild-type strain, indicating that compared with wild-type strain, ΔluxS strain significantly reduced virulence. The virulence of the complementation mutant strain C-luxS was restored, with generally the same LD₅₀ value as the wild-type strain. Similar to FK6768, the LD₅₀ value of ΔluxS strain of K. pneumoniae FK563 was 14.24-fold compared to that of the wild-type strain, indicating significantly attenuated virulence of ΔluxS strain. The virulence of the complementation mutant strain C-luxS was partially restored, with an LD₅₀ value generally the same as that of the wild-type strain (Table 7).

TABLE 7.

LD₅₀ and LD₉₀ values of wild-type, ΔluxS, and C-luxS K. pneumoniae strains in mice

K. pneumoniae isolates	Strains	Challenge dose (CFU)	Percent of dead mice (%)	Value of LD50 CFU	Fold changea	Value of LD90 CFU	Fold changea
FK1979	Wild-type	2.23 × 103	20 (1/5)	9.26 × 103	1	2.52 × 104	1
		5.11 × 103	40 (2/5)
		1.01 × 104	60 (3/5)
		2.04 × 104	80 (4/5)
		4.60 × 104	100 (5/5)
		1.04 × 105	100 (5/5)
	ΔluxS	0.98 × 104	0 (0/5)	9.22 × 104	9.96	3.83 × 105	15.20
		2.22 × 104	0 (0/5)
		5.10 × 104	0 (0/5)
		1.32 × 105	20 (1/5)
		5.12 × 105	60 (3/5)
		0.95 × 106	100 (5/5)
	C-luxS	1.08 × 103	0 (0/5)	1.5 × 104	1.62	7.35 × 104	2.92
		2.17 × 103	0 (0/5)
		5.80 × 104	20 (1/5)
		1.25 × 104	40 (2/5)
		5.14 × 104	100 (5/5)
		1.09 × 105	100 (5/5)
FK6768	Wild-type	1.12 × 106	20 (1/5)	1.99 × 107	1	7.94 × 107	1
		1.03 × 107	40 (2/5)
		2.63 × 107	60 (3/5)
		5.60 × 107	80 (4/5)
		1.11 × 108	100 (5/5)
		1.25 × 109	100 (5/5)
	ΔluxS	1.09 × 106	0 (0/5)	9.80 × 107	4.92	2.71 × 108	3.41
		0.95 × 107	0 (0/5)
		2.07 × 107	0 (0/5)
		4.90 × 107	20 (1/5)
		0.98 × 108	60 (3/5)
		1.04 × 109	100 (5/5)
	C-luxS	1.07 × 106	0 (0/5)	2.20 × 107	1.11	8.33 × 107	1.05
		0.97 × 107	20 (1/5)
		2.12 × 107	60 (3/5)
		5.10 × 107	80 (4/5)
		1.08 × 108	100 (5/5)
		1.15 × 109	100 (5/5)
FK563	Wild-type	0.98 × 106	0 (0/5)	2.05 × 107	1	1.14 × 108	1
		1.06 × 107	20 (1/5)
		2.21 × 107	40 (2/5)
		5.20 × 107	60 (3/5)
		1.13 × 108	100 (5/5)
		1.25 × 109	100 (5/5)
	ΔluxS	1.31 × 106	0 (0/5)	2.92 × 108	14.24	3.18 × 108	2.79
		0.99 × 107	0 (0/5)
		2.02 × 107	0 (0/5)
		4.90 × 107	0 (0/5)
		1.08 × 108	40 (2/5)
		1.21 × 109	100 (5/5)
	C-luxS	1.42 × 106	0 (0/5)	4.33 × 107	2.11	9.28 × 107	0.81
		1.25 × 107	0 (0/5)
		2.43 × 107	40 (2/5)
		5.10 × 107	60 (3/5)
		1.22 × 108	100 (5/5)
		1.27 × 109	100 (5/5)

^a Fold change normalized to the wild-type strain (wild-type).

Survival curve assay on mice

Survival curve assay was used to further assess the effects on bacterial pathogenicity of the luxS gene in the mice infection model. A preliminary experiment revealed that the doses corresponding to 10⁸ cells of the wild-type strain of K. pneumoniae FK6768 or FK563 were required to cause 100% lethality in this infection model (Fig. 1E). We then compared the pathogenicity of the wild-type and each mutant at two different infecting doses (10⁸ and 10⁷ cells). As expected, all mice infected with 10⁸ cells of the wild-type strains died within 48 h post-infection. In contrast, at the same infecting dose, the luxS mutants only caused lethality in 40% and 50% of mice. Similarly, 40% and 80% lethality were observed in mice infected with 10⁷ cells of wild-type strains, while the corresponding luxS mutants were almost avirulent (0% and 20% of mortality for FK6768 and FK563, respectively) (Fig. 2A). These results suggested that the challenge with the luxS mutant had a less deleterious effect on mice than the wild-type strains. Taken together, these results demonstrate that luxS contributed to K. pneumoniae pathogenicity in mice models of infection.

Fig 2.

LuxS contributes to virulence in a mouse model of bacteremia. ICR mice were intraperitoneal (i.p.) inoculated with 2 × 10⁶ CFUs of the wild-type (WT) or ΔluxS strain of K. pneumoniae FK6768 or FK563. (A) Growth curve of mice infected with the indicated strain. Differences in mortality were compared to the WT group using the Mantel-Cox log-rank test. ns, not significant, *, P < 0.05, ***, P < 0.001. (B) Mice were euthanized at 24 h postinoculation (hpi), and the blood, heart, liver, spleen, lungs, and kidneys were homogenized and plated for bacterial enumeration. The dotted line indicates the limit of detection, and symbols on the dotted line indicate CFU counts that were below the limit of detection. These data are from an individual representative experiment. The Mann-Whitney test was used for statistical analyses comparing each mutant to the WT. ns, not significant, *, P < 0.05. (C) Expression of inflammatory factors TNF-α, IL-1β, and IL-6 of liver, spleen, and lungs of ICR mice infected with wild-type, luxS mutant strains, and PBS (blank control) detected by qPCR and calculated by relative expression 2^−△△CT. Results are expressed as the relative differences of ΔluxS/WT. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. (D) Pathological characterization of liver, spleen, and lungs tissues of mice infected with WT and luxS mutant strains. Histologic evidences were examined by hematoxylin and eosin (H&E) staining and observed under a microscope (magnification, ×200) in mice infected with WT and ΔluxS 24 h after. The scale bars represent 100 µm. The Mann-Whitney test was used for statistical analyses of pathological scores between the WT and ΔluxS. ns, not significant, *, P < 0.05, **, P < 0.01. (E) Representative flow cytometry plots showing CD45⁺ CD11b⁺ Ly6C⁺ Ly6G⁺ neutrophil and monocyte recruitment. CD45⁺ CD11b⁺ immune cells were gated from white cells of blood and then distinguished by using Ly6C as the abscissa and Ly6G as the ordinate.

Bacterial burden, pathology, and inflammation level of tissues

Based on animal survival outcomes above, we determined that luxS is important for K. pneumoniae pathogenicity. To determine whether reduced survival of mice infected with luxS deletion mutant is associated with changes in bacterial loads, CFUs (colony-forming units) were quantified from blood, heart, liver, spleen, lung, and kidney samples collected 24 h after i.p. inoculation of approximately 1 × 10⁷ CFUs per strain in mice (all animals from both groups were alive to avoid survivor bias). The bacterial loads in blood, heart, liver, spleen, lung, and kidney were significantly higher in mice infected with wild-type strains than those in mice with luxS deletion mutants, demonstrating that the increased mortality of mice with wild-type is associated with the enhanced virulence of K. pneumoniae due to luxS (Fig. 2B). As shown in Fig. 2C, knockout of luxS gene weakened the pro-inflammatory ability of FK6768 and FK563 (Fig. 2C). Pathological scores after exposure revealed that luxS mutants had less histologic evidence of liver, spleen, and lung. The results showed that luxS gene knockout attenuated the tissue damage ability of FK6768 and FK563, consistent with the decreased pathogenicity (Fig. 2D). Cellular response to infection in blood determined by flow cytometry showed that the rates of Ly6G^high Ly6C^high cells increased in mice infected with ΔluxS strain compared with that of the mice infected with the wild-type (Fig. 2E). These data suggested that luxS contributed to the virulence of regular-mucoid and nonmucoid K. pneumoniae strains.

LuxS regulates the pathogenicity of hypermucoid K pneumoniae FK1979 in a density-dependent manner

The above in vivo virulence studies of FK6768 and FK563 are consistent with the reduced expression of bacterial virulence relative genes in their logarithmic growth phase shown by transcriptome sequencing, indicating that LuxS might promote virulence and pathogenicity of K. pneumoniae to a certain extent; interestingly, however, for FK1979 in its logarithmic growth phase, there was no significant difference in virulence-related gene expression between wild-type and knockout strains. Since research has shown that LuxS plays a phenotypic regulatory role via sensing bacterial cell density, we next explore whether LuxS regulates the pathogenicity of FK1979 by comparing the pathogenic capacity in mice between luxS mutation and wild-type strains at their two different growth stages: early low-density growth and late high-density growth in the subsequent study of FK1979.

In our study, both wild-type and luxS knockout strains of FK1979 were cultured under identical conditions, cultivated to early low-density growth phase and late high-density growth phase, and then used to infect two groups of mice (with each group consisting of six individuals) with the same CFU to assess the impact of luxS mutation on bacterial virulence. The results revealed that, for bacteria cultured to the early logarithmic growth phase, the mortality rate in mice infected with the knockout strain, at the same CFU, showed a slight increase compared to mice infected with the wild-type strain, albeit without significant statistical differences. In contrast, for bacteria cultured to the late high-density growth phase, the mortality rate in mice infected with the knockout strain, at the same CFU, exhibited a significantly reduced mortality rate compared to mice infected with the wild-type strain (Fig. 3A). Further bacterial load experiments using ΔluxS mutant and wild-type of FK1979 indicated that at 24 hpi, the bacterial loads of the tissues of mice reached to 8–9 logs from the initial inoculum of 7 logs, implying FK1979 could proliferate in vivo. For bacteria cultured to the late high-density growth phase, the bacterial load of the wild-type-infected mice was 1–2 logs higher than those of ΔluxS mutant infected mice. However, for the bacteria cultured to the early logarithmic growth phase, the bacterial load of mice infected with ΔluxS mutant were 1–2 logs higher than those of the mice infected with wild-type (Fig. 3B). Consistent with the changing trend of the bacterial load, the IL-6 of the mice infected with ΔluxS mutant were significantly lower than those of the wild-type infected mice for bacteria cultured to the late high-density growth phase; while for the bacteria cultured to the early logarithmic growth phase, the IL-6 of mice infected with ΔluxS mutant were significantly higher than those of the mice infected with wild-type (Fig. 3C). Pathological scores after infection revealed that luxS mutants had more histologic evidence of liver, kidneys, and lungs than wild-type at the low-density state, but luxS mutants had less histologic evidence of liver, kidneys, and lungs than wild-type at the high-density state (Fig. 3D). Cellular response to infection in blood determined by flow cytometry showed that the rates of Ly6C^lowLy6G⁺ and Ly6C^high cells in mice infected with ΔluxS strain were decreased when compared with that of the mice infected with the wild-type at the high-density phase; while the rates of Ly6C^lowLy6G⁺ and Ly6C^high cells increased at the low-density phase (Fig. 3E). Taken together, these results demonstrate that LuxS contributed density-dependently to virulence of hypermucoid K. pneumoniae in a mouse model of infection.

Fig 3.

LuxS contributes to virulence in a mouse model of bacteremia. ICR mice were intraperitoneal (i.p.) inoculated with 2 × 10³ CFUs of the wild-type (WT) or ΔluxS strain of K. pneumoniae FK1979. (A) Growth curve of mice infected with the indicated strain. Differences in mortality were compared to the WT group using the Mantel-Cox log-rank test. ns, not significant, *, P < 0.05. (B) Mice were euthanized at 24 h postinoculation (hpi), and the blood, heart, liver, spleen, lungs, and kidneys were homogenized and plated for bacterial enumeration. The dotted line indicates the limit of detection, and symbols on the dotted line indicate CFU counts that were below the limit of detection. These data are from an individual representative experiment. The Mann-Whitney test was used for statistical analyses comparing each mutant to the WT. ns, not significant, *, P < 0.05. (C) Expression of inflammatory factors TNF-α, IL-1β, and IL-6 of liver, spleen, and lungs of ICR mice infected with wild-type, luxS mutant strains, and PBS (blank control) detected by qPCR and calculated by relative expression 2^−△△CT. Results are expressed as the relative differences of ΔluxS/WT. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. (D) Pathological characterization of liver, kidneys, and lungs of mice infected with and luxS mutant strains. Histologic evidences were examined by hematoxylin and eosin (H&E) staining and observed under a microscope (magnification, ×200) in mice infected with WT and ΔluxS 24 h after. The bars represent 100 µm. The Mann-Whitney test was used for statistical analyses of pathological scores between the WT and ΔluxS. ns, not significant, *, P < 0.05, **, P < 0.01. (E) Representative flow cytometry plots showing CD45⁺ CD11b⁺ Ly6C⁺ Ly6G⁺ neutrophil and monocyte recruitment. CD45⁺ CD11b⁺ immune cells were gated from white cells of blood and then distinguished by using Ly6C as the abscissa and Ly6G as the ordinate.

luxS affects the interactions with host cells, biofilm formation, and serum resistance of K. pneumoniae, as well as the LPS and CPS expression

To further clarify the reasons for the different effects of luxS on the in vivo pathogenicity of K. pneumoniae, we next conducted in vitro experiments to identify the impacts of luxS on the interactions with host cells, biofilm formation, and serum resistance. Because of the reduced lethality and bacterial burden of the ΔluxS mutants in mice, we wanted to determine if these strains had altered interactions with mice macrophage cells (RAW-264.7 cells). The wild-type and ΔluxS mutant strains were co-incubated with RAW-264.7 cells and their adhesion and invasion into the cell were assessed. ΔluxS mutants of FK1979 exhibited lower adhesion and phagocytosis rates at the low-density growth state and higher rates at the high-density state. FK6768 displayed the same trend as FK1979. For FK563, however, ΔluxS mutants were more adherent and more readily phagocytosed than the wild-type at both the low- and high-density growth state (Fig. 4A1). Phagocytosis analysis by flow cytometry also showed the same trends of the difference as the bacterial counting (Fig. 4A2). To determine the function of luxS in virulence, a cytotoxicity assay was performed on RAW-264.7 cells to evaluate in vitro toxicity of K. pneumoniae FK1979, FK6768, FK563 strains, and their ΔluxS mutants. After infection, the release of LDH was considered to be an indicator of host cell-membrane integrity and cell viability in the cytotoxicity assay. When RAW-264.7 cells were infected with the ΔluxS strains, the cytotoxicities of ΔluxS strains of FK6768 and FK563 were less than those of the wild-type strains. However, the cytotoxicity of the ΔluxS FK1979 strain at the early low-density growth phase was enhanced, which indicated that the illusion of reduced adhesion phagocytosis caused by cell death can be excluded, indicating that it is indeed the reduced adhesion and phagocytosis of the FK1979 ΔluxS strain (Fig. 4B). TNF-α, IL-1β, and IL-6 are pro-inflammatory cytokines that play key roles in initiating and regulating the inflammatory response. During a bacterial infection, these cytokines are released by immune cells as part of the early response to infection. Detection of elevated levels of these cytokines indicates the activation of the immune system and the presence of an inflammatory state. There was no significant difference in the expression of inflammatory factors TNF-α, IL-1β, and IL-6 of RAW264.7 cells infected with wild-type and ΔluxS mutant of FK1979 at their early low-density growth state, while IL-6 was significantly decreased in ΔluxS group at the late high-density growth phase. For FK6768 and FK563, however, the inflammatory factors of cells infected with ΔluxS mutant were remarkably higher than that of the wild-type (Fig. 4C1). TLR4-MyD88-NFκB pathway plays a crucial role in the immune response to bacterial pathogens. TLR4 is a pattern recognition receptor that recognizes specific molecular patterns associated with bacteria, such as lipopolysaccharide (LPS), thereby initiating downstream signaling pathways, including MyD88 and NFκB. The activation of this pathway ultimately activates immune cells to release inflammatory factors, such as TNF-α, IL-1β, and IL-6, to counteract the infection. The TLR4-MyD88-NFκB pathway expression level represents the impact of bacterial infection on the host immune system. The expression of the TLR4-MyD88-NFκB pathway of cells infected with ΔluxS strains was strikingly higher than that of wild-type for FK6768 and FK563, while there was no significant difference between ΔluxS and wild-type strains for FK1979 (Fig. 4C2). According to Fig. 4D, the serum resistance of ΔluxS strain in FK6768 and FK1979 was markedly enhanced compared with its wild-type strain at the early low-density growth state, but it was the opposite at the high-density phase. Nonetheless, the serum resistance of the ΔluxS strain of FK563 did not differ from its parental strain (Fig. 4D). To investigate the difference in biofilm formation between the wild-type and the △luxS mutant strains, the biofilm formation was quantified at different time points using a microtiter plate crystal violet (CV) staining and a confocal laser scanning microscope (CLSM) method. The ΔluxS strains formed considerably fewer biofilms than wild-type strains during the entire observation period (Fig. 4E), which complied with the transcriptomics study that the expression levels of the biofilm-related genes, including Fim and Mrk genes, of FK6768 and FK563 were significantly decreased in their ΔluxS mutant strains (Table 5), which might result in significant decrease of biofilm formation of K. pneumoniae ΔluxS mutant. Fim and Mrk genes were both found in bacteria and were involved in the synthesis of fimbriae or pili, which are hair-like structures on the surface of bacterial cells that play a role in adhesion to host tissues and biofilm formation. Further qRT-PCR detecting the gene expression in the early and late growth state showed that the CPS expression of △luxS increased at the early low-density growth phase and decreased at the late high-density growth phase, compared with the wild-type in FK1979; while the expression of LPS of △luxS increased compared with the wild-type of FK6768 and FK563 (Fig. 5). The gene expression results indicated that luxS may regulate the expression of bacterial CPS, LPS, biofilm, and other virulence genes in a bacterial density-dependent manner to affect bacterial virulence and host immune response to bacteria, thus affecting the outcome of host infection. All in all, our results illustrated that the impact of LuxS on the pathogenicity of K. pneumoniae is highly correlated with the mucoid phenotype and growth stage of the strains, which might explain the different outcome observed in mice models of infection.

Fig 4.

luxS gene affects the interactions with host cells, biofilm formation, and serum resistance of K. pneumoniae. (A1) Host cell adhesion and phagocytosis of K. pneumoniae FK1979, FK6768, FK563 ΔluxS, and their parental strains in RAW-264.7 cells via bacteria counting by plating. Statistical analyses were performed using the two-way ANOVA between the WT and ΔluxS. ns. not significant; **P < 0.01 and ***P < 0.001. (A2) Phagocytosis analysis by flow cytometry. WT (upper panels) and ΔluxS (lower panels) following labeled by TRITC were phagocytosis by activated RAW-264.7 cells. (B) The impact of luxS deletion of K. pneumoniae FK1979, FK6768, and FK563 on the cytotoxicity was assessed by LDH leakage assay. The values are presented as means ± SD of triplicates. The Mann-Whitney test was used to assess the significance of differences between the WT and ΔluxS. ns, not significant, *, P < 0.05. (C1) Expression of inflammatory factors TNF-α, IL-1β, and IL-6 of RAW264.7 cells infected with wild-type, luxS mutant strains, and PBS (blank control) detected by qPCR and calculated by relative expression 2^−△△CT. Results are expressed as the relative differences of ΔluxS/WT. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001. (C2) TLR4-MyD88-NFκB pathway expression difference via western blot. (D) Serum complement-mediated killing of K. pneumoniae FK1979, FK6768, and FK563 isolates. Based on the results of the preliminary experiment, the initial bacterial counts of K. pneumoniae FK1979, FK6768, and FK563 were approximately 1 × 10⁶, 1 × 10⁸, and 1 × 10⁸ CFU/mL, respectively. The values are presented as means ± SD of triplicates. Repeated measures analysis of variance (ANOVA) was employed to assess the significance of differences between the WT and ΔluxS at the same growth state. ns, not significant, *, P < 0.05. (E1) Biofilm formation of K. pneumoniae FK1979, FK6768, FK563 ΔluxS, and their parental strains was quantitatively detected by crystal violet staining at different growth time points. Data are expressed as the ratio of OD_570/595nm of three independent experiments. Student’s t test was used to assess the significance of differences between the WT and ΔluxS within the same time point. ns, no significance, *, P  < 0.05. (E2) The biomass and structure of biofilm at 24 h was observed by confocal laser scanning microscope (CLSM).

Fig 5.

Expression of CPS and LPS genes of luxS mutant relative to wild-type of K. pneumoniae FK1979 (A), FK6768 (B), and FK563 (C), detected by qPCR and calculated by relative expression 2^−△△CT. ns, not significant, *, P < 0.05, **, P < 0.01, ***, P < 0.001.

DISCUSSION

LuxS/AI-2 has been proven as a critical QS system that influences various physiological functions such as bacterial growth, biofilm formation, virulence, and metabolism in multiple bacteria (7, 13), but the role of LuxS in these processes has remained poorly addressed in K. pneumoniae. Our previous work identified the relationships among LuxS/AI-2 QS, biofilm formation, and gene expression of outer-membrane components in K. pneumoniae (14). Characterizing the regulatory role of luxS on the physiological functions of K. pneumoniae strains will improve our understanding of this important pathogen.

High genetic heterogeneity of EPS biosynthesis endows K. pneumoniae with different mucoid phenotypes, which complicated the study of their pathogenicity and the corresponding regulatory mechanisms (3, 15). The hallmark of K. pneumoniae is the production of CPSs, leading to bacterial colonies that typically exhibit a certain degree of mucosity. Many K. pneumoniae strains are smooth, and cream-colored and appear as a regular-mucoid phenotype with a negative string test. However, some strains look shiny and cream-colored on blood agar plates, and showed a hypermucoid phenotype with a positive string test. In addition, some other strains look dry, translucent in appearance, and have a comparably smaller colony size (16). K. pneumoniae can be divided into hypermucoid, and nonmucoid, according to the difference of the CPS production, which usually correspond to hypervirulent and low-virulent strains. The overwhelming majority of strains, however, may contain a moderate amount of CPS and virulence genes, characterized by a certain mucosity with a regular-mucoid phenotype, but lower virulence than the hypervirulent type (16). The bacterial characteristics show that the three clinical K. pneumoniae strains used in the current study represent the hypervirulent hypermucoid, low-virulent regular-mucoid, and nonmucoid phenotypes. Some previous studies have implied that the pathogenicity and the corresponding host immune response are significantly different between the hypermucoid, regular-mucoid, and nonmucoid K. pneumoniae (17). We studied the effects of LuxS on bacterial virulence and pathogenicity in these three representative strains with different mucoid phenotypes, which can provide a more comprehensive theoretical basis for target screening for clinical treatment of K. pneumoniae infections. As the transcriptome analysis of wild-type K. pneumoniae and its isogenic luxS knockout mutant shows, luxS deletion reduced the expression of virulence gene and biofilm-related genes compared with the wild-type strains in regular-mucoid and nonmucoid K. pneumoniae, while no significant difference was observed in hypermucoviscous strain. This diversity triggered us to clarify the strain-dependent regulatory mode of luxS on the pathogenicity of K. pneumoniae.

Experiments in vivo can best reflect the virulence and pathogenicity of bacteria. We used mice infection models to conduct experiments. The results showed that knocking out the luxS gene of the regular-mucoid and nonmucoid K. pneumoniae could significantly reduce their pathogenicity in mice. Further mechanistic studies indicate that the deletion of the luxS gene in regular-mucoid and nonmucoid K. pneumoniae renders them more susceptible to host cell phagocytosis and serum killing. Additionally, their virulence toward host cells and biofilms formation are significantly reduced. Interestingly, luxS knockout upregulated pro-inflammatory factors such as IL-1β, IL-6, and TNF-α through activation of TLR4-MyD88-NFκB in macrophage cells. Since classical K. pneumoniae has been reported to evade host immunity through immunosuppressive mechanisms (18), the knockout strain may potentially enhance host protective immunity, thereby rendering it more susceptible to clearance by the host. LPS is one of the main pathogen-associated molecular patterns that can be recognized by TLR4, which leads us to hypothesize that luxS knockout promotes the expression of LPS. The increased expression of LPS of luxS knockout in the regular-mucoid and nonmucoid K. pneumoniae, in line with the findings of De Araujo et al. (19), further supported the hypothesis. Infection outcome is determined by both bacterial virulence and the host immune response (20). These findings elucidate why luxS gene knockout of regular-mucoid and nonmucoid K. pneumoniae increases the survival rate and LD₅₀ of mice, and decreases the bacterial load in tissues and organs, as well as stimulates host immune response. The combined effects of bacteria and the host immune result in a more favorable clinical outcome in animals for luxS knockout than wild-type in the regular-mucoid and nonmucoid K. pneumoniae.

The QS regulates bacterial physiological activities in response to bacterial cell density (8–10). The absence of detectable differences in RNA expression profiles between the wild-type and luxS knockout mutant strains at their logarithmic growth phase in hypermucoviscous K. pneumoniae prompts us to investigate the potential regulatory effects of LuxS on bacterial pathogenicity and its relationship with bacterial cell density. Therefore, we subsequently investigated the phenotypic regulatory role of LuxS on hypermucoviscous K. pneumoniae FK1979 under both high and low-density conditions, to further elucidate its relationship with bacterial cell density. It was revealed that in hypermucoid K. pneumoniae, LuxS regulated bacterial virulence and pathogenicity in a bacterial growth state-dependent manner, and LuxS inhibited its virulence and pathogenicity at the early low-density growth stage but promoted them at the late high-density growth stage. Further mechanistic studies indicate that the luxS mutant were less susceptible to host cell phagocytosis and serum killing, as well as exhibiting higher cytotoxicity, than the wild-type at their early low-density growth stage; whereas the opposite was observed at the late high-density growth stage. One prominent characteristic of hypermucoviscous K. pneumoniae, in contrast to the classical type, is the markedly elevated production of CPS. CPS is a thick outer layer of complex polysaccharides that surrounds the bacterial cell wall, which protects K. pneumoniae from host immune system recognition and clearance (21–23). The CPS can act as a barrier, preventing phagocytosis, opsonization, and complement activation, making hypermucoviscous K. pneumoniae more resistant to host defense mechanisms (21–23). The CPS is known to suppress phagocytosis, and unencapsulated K. pneumoniae strains are more easily phagocytosed and killed by phagocytic leukocytes than encapsulated strains (21). Furthermore, the presence of CPS is directly related to K. pneumoniae virulence, resulting in strains that produce capsules being more invasive and cause more severe infections than non-capsulated strains (21, 23, 24). These roles of CPS, combined with the above results of mechanistic studies, promote us to speculate that LuxS regulates CPS in a density-dependent manner in hypermucoviscous K. pneumoniae. CPS gene expression detection showed that luxS mutant promoted CPS gene expression at its early low-density growth phase and inhibited CPS gene expression at its late high-density growth phase in hypermucoviscous K. pneumoniae, further supporting this hypothesis. However, it is interesting that we did not observe significant differences in the ability to stimulate TLR4-MyD88-NFκB and inflammatory factors of host between the wild-type and knockout strains. This lack of distinction may be attributed to the TLR4-MyD88-NFκB pathway primarily sensing LPS, while LuxS in FK1979 predominantly influences CPS. The profound impact of hypermucoviscous K. pneumoniae on host immunity primarily arises from its ability to induce rapid and overwhelming recruitment of neutrophils, resulting in destructive host immune responses (24). Analysis of the immune cell populations in infected mice revealed the proportion of cell subpopulations indicative of this destructive immune response increased in the knockout strain under low-density growth conditions, whereas they decreased under high-density growth conditions. Combined with the conclusion above that LuxS inhibits LPS, we speculate that LuxS may also inhibit LPS of hypermucoviscous K. pneumoniae. However, as the CPS is external to the LPS, the interactions between hypermucoviscous K. pneumoniae and the host might be preferentially determined by the CPS. Alternatively, the impact of LuxS on the virulence and pathogenicity of hypermucoviscous K. pneumoniae may be primarily manifested through alterations in CPS expression.

Biofilm formation is regulated by quorum sensing in many bacteria (25). Our results are similar to several other studies, which have demonstrated that LuxS promotes biofilm production. Biofilms are considered a developmental process as they are formed in steps that require intercellular signaling. LuxS/AI-2 QS has been reported as important for full biofilm formation (26). Thus, it is possible that LuxS possibly plays a general regulatory role in the pathogenicity of K. pneumoniae, by inhibiting LPS, suppressing CPS production at the early low-density growth phase, and promoting CPS and biofilm formation at the late high-density stage. In the case of hypermucoviscous K. pneumoniae, it exhibits both early inhibition and late-stage promotion, which may explain the not significantly differential expression observed in the log-mid phase transcriptome sequencing data, which was in line with some other studies (27).

All in all, our results demonstrated strain-dependent variation in the contributions of LuxS to the virulence and pathogenicity of regular-mucoid, nonmucoid, and hypermucoid K. pneumoniae. Our findings provide new insights into the important contribution of QS system LuxS/AI-2 to the networks that regulate the pathogenicity of K. pneumoniae and proved the regulatory pattern of LuxS/AI-2 QS on K. pneumoniae was growth phase-dependent. We believe that our data will facilitate our understanding of regulatory mechanisms of LuxS/AI-2 QS on the pathogenicity of K. pneumoniae under the background of their genetic heterogeneity and help develop new strategies for diminished bacterial virulence within the clinical K. pneumoniae population.

MATERIALS AND METHODS

Bacterial strains, plasmids, primers, and culture conditions

The bacterial strains used in this study were isolated from the First Affiliated Hospital of Wenzhou Medical University. The ΔluxS mutants of K. pneumoniae FK1979, FK6768, and FK563 were constructed using the λ red homologous recombination method and reserved in our laboratory (14). The bacterial culture process before the experiment was carried out according to the same procedure, i.e., after overnight cultivation of single bacterial colonies, a 1:1,000 subculture was prepared in LB broth. The early low-density growth state was referred to the 3 h after the initial inoculation and the late high-density growth state was referred to the 12 h after the initial innovation. The concentration of the bacteria (CFU/mL) used in each experiment was determined by preliminary McFarland turbidimetry and accurate colony counting.

RNA sequencing and bioinformatics analysis

The wild-type, ΔluxS strains were grown overnight (8–10 h) at 37°C, and then 1 mL of the cultures were added into 100 mL of LB to reach the mid-exponential phase. Then, total RNA was extracted using RNAprep Pure Cell/Bacteria Kit (Tiangen, Beijing, China), and the entire sequencing was conducted by the BGI Company. Each sample was performed using three biological replicates. The expression of Unigene was calculated by RPKM method (Reads Per kb per Million reads). The relative expression levels were estimated by normalizing the transcript level in wild-type as 1. The relative expression of the indicated genes was calculated by the 2^−ΔΔCt method and presented as fold change of ΔluxS mutant relative to wild-type in Tables 3–6.

Bacterial growth curve determination

The growth kinetics of the strains were determined by monitoring culture optical density (OD at 600 nm) for 24 h at 37°C (28). Briefly, overnight cultures were re-cultured 1:100 in 5 mL of fresh LB and incubated with shaking at 37°C and 180 rpm to logarithmic phase and the OD₆₀₀ values were detected every 2 h at the indicated time points by transferring in triplicates into a 96-well microtiter plate (Corning, USA). The tests were carried out independently in triplicate.

Assessment of CPS production and mucoviscosity

The string test was qualitatively detected for HMV (>5 mm). The optical density at 600 nm (OD₆₀₀) of culture supernatants was measured following low-speed centrifugation (1,000 × g for 5 min) to assess the mucoviscosity. Acidic polysaccharides were extracted and measured to quantify CPS as described previously (29). Briefly, 500 µL of the bacterial culture of log-phase was mixed with 100 µL of 1% ZWITTERGENT 3–14 detergent in 100 mM citric acid, followed by incubation at 50°C for 20 min. The cells were then pelleted (14,000 × g for 2 min). About 300 µL of the supernatant was aspirated and mixed with 1.2 mL of absolute ethanol, incubated at 4°C for 20 min, and then centrifuged for 5 min at 14,000 × g. The pellet was dried and resuspended in 200 µL of distilled water. Then, 1.2 mL of 12.5 mM sodium tetraborate in sulfuric acid was added and incubated for 5 min at 100°C, followed by incubation on ice for 10 min. Thereafter, a 20-µL volume of 0.15% 3-phenyl phenol in 0.5% NaOH was added. After a 5-min incubation at room temperature, the absorbance at 520 nm was measured.

Serum resistance assay

The bacterial resistance to serum bactericidal activity was determined in pooled normal mouse serum. Briefly, 1 × 10⁶, 1 × 10⁸, and 1 × 10⁸ CFU/mL of log-phase bacteria for K. pneumoniae FK1979, FK6768, and FK563 were mixed with serum at a 1:3vol/vol ratio, respectively. The mixture in a final volume of 1mL was incubated for 3 h at 37°C; and at 0, 1, 2, and 3 h intervals, 100 µL of aliquots was removed, diluted, and cultured on LB agar for colony enumeration.

Mice infection model

All animal experiments in this study were approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (wydw2021-0023) and performed according to the relevant guidelines and regulations. Six- to 8-week-old female ICR mice were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd, China. All mice were housed in individually filtered cages and received unlimited sterile food and water. Mice were anesthetized with isoflurane throughout the experimental procedures and sacrificed by inhaling CO₂ to minimize suffering after the experiment.

Determination of the LD₅₀ in mice was first carried out to evaluate the virulence of the tested K. pneumoniae clinical isolates FK1979, FK6768, and FK563 and their luxS mutants. Female ICR mice (aged 6–8 weeks, five mice per group) were intraperitoneally injected with 200 µL of bacterial dilutions. Deaths within 7 days after inoculation were monitored. The LD₅₀ was estimated by non-linear regression analysis using GraphPad software. For the assessment of bacterial virulence of K. pneumoniae FK1979, FK6768, and FK563, fresh bacterial cultures of the strains were washed, and resuspended in PBS to get final concentrations of approximately 1.0 × 10⁴, 1.0 × 10⁵, 1.0 × 10⁶, 1.0 × 10⁷, 1.0 × 10⁸, 1.0 × 10⁹, and 1.0 × 10¹⁰ CFU/mL. The bacterial concentration infected was calculated by the CFU counting. For the assessment of bacterial virulence of wild-type strain, mutant strain ΔluxS, and complemented strain C-luxS, fresh bacterial cultures of the strains were washed, and resuspended in PBS to get six final concentrations of approximately 1.0 × 10⁴–10⁷, 1.0 × 10⁷–10¹⁰ CFU/mL (based on the LD₅₀ of specific strain).

Survival curve of the mice assay was carried out as described previously (30). In the determination of survival curves in mice, 10 mice were used as a sample population for each bacterial concentration. The bacterial concentration infected was calculated by CFU. Infection doses of bacteria were derived from the MLD value determined through the LD₅₀ determination assay. 10⁵, 10⁸, and 10⁸ CFUs of the wild-type of FK1979, FK6768, and FK563 resulted in 100% death within 48 h, whereas 10⁴, 10⁷, and 10⁷ CFUs resulted in a sufficient mortality rate at an appropriate observation interval. The two concentrations of each of the three bacteria were tested. Death rates were recorded for 7 days. The log-rank test was performed using GraphPad Prism 8.

Bacteriological, histopathological, and inflammation examination

To determine the viable bacteria in mice organs, a total of 12 female ICR mice were randomly and averagely assigned to three groups (wild-type and ΔluxS strain infection group, and control group without infection, n = 4) and used for assessing the presence of viable bacteria in infected mice organs for each K. pneumoniae isolate. The experimental mice were injected i.p. with 200 µL of wild-type and ΔluxS strains (5 × 10³, 2 × 10⁷, and 2 × 10⁷ CFUs, for K. pneumoniae FK1979, FK6768, and FK563, respectively), and the control mice were injected with 200 µL of PBS. After infection for 48 h, tissue samples of blood, liver, spleen, lung, and kidney were collected and homogenized. The liver, spleen, and lungs were suspended in 1 mL of cold sterile PBS and homogenized using a homogenizer (Retsch, China) at 4°C. The specimen was serially diluted and plated on LB agar to measure the CFU per tissue, followed by incubation at 37°C for 16–20 h. The number of colonies was counted and presented as CFU/g or CFU/mL. Three plates were used at every dilution and each experiment was performed in triplicate. For histopathological analysis, tissues were fixed in 10% neutral buffered formalin for 48 h, and the paraffin-embedded sections, cut into 5 µm sections, were stained with hematoxylin-eosin (H&E; Sigma). Light microscopy (Nikon Eclipse 55i, Japan) was used to obtain the images. Scores of 0, 1, 2, 3, and 4 were assigned to the four indicators of neutrophils, necrosis, vascularlesion, and granuloma, respectively, and the total score was recorded as the pathological score of each group. For the inflammatory cytokines test, the expression levels of TNF-α, IL-6, and IL-1β in the tissues were determined by using RNA extraction and qPCR method.

Flow cytometry

For flow cytometry staining, approximately 10⁷ cells were counted from red blood cell (RBC)-lysed blood samples. Each sample was incubated with an anti-CD16/32 antibody cocktail (Fc Block; BioLegend, San Diego, CA) for 5 min at room temperature. Then, cellular markers were surface labeled by adding a 1:200 dilution of each antibody for 20 min at 4°C. The following antibodies/dyes were used to immunophenotype the cellular infiltrates: anti-CD45-APC (clone 30-F11), anti-CD11b-BV605 (clone M1/70), Ly-6C-PB (clone HK1.4), and Ly-6G-PE (clone 1A8) from BioLegend (San Diego, CA). Flow cytometry data were analyzed using FlowJo version 9.9.4 software (TreeStar, LLC). Cell populations were determined after gating on CD45⁺ leukocytes, Ly6G⁺/CD11b⁺ neutrophils, and Ly6C⁺ CD11b⁺ inflammatory monocytes.

Cell adhesion and invasion assays

Murine macrophage cells RAW 264.7 (Procell Life Science and Technology Co., Ltd., China) were cultured in DMEM medium which was supplemented with 10% heat-inactivated fetal bovine serum (HIFBS; GIBCO, Life Technologies) and 100 U/mL penicillin + 100 µg/mL streptomycin (Gibco, Life Technologies, Waltham, MA) at 37°C in a 5% CO₂ incubator. For the adhesion experiment, RAW 264.7 cells were seeded at an initial density of 5 × 10⁵ cells per well in 24-well flat-bottom microplates (Nest, USA). Overnight cultures of each strain were sub-cultured to the logarithmic growth phase and were adjusted to 0.5 McF. The culture medium was aspirated out and replaced with fresh DMEM which contained the indicated CFU of bacteria with an optimal multiplicity of infection (MOI) MOI = 20:1 for FK1979; 50:1 for FK6768 and FK563, for 3 h in a CO₂ incubator. For invasion assays, polymyxin B (Kangtai, China) was used to kill the extracellular bacteria of RAW264.7. After 3 h of bacterial infection, 1 mL of 2 µg/mL polymyxin B was introduced to the 24-well plate for 1 h. After washing with PBS four times, the bacteria were released with 1 mL of 0.1% Triton X-100 solution. The bacteria were 10-fold serial diluted and plated onto LB agar for colony count. The adhesion and invasion rates were calculated according to the enumeration of the bacterial CFUs of each strain. After the same phagocytosis process was carried out the bacteria were labeled with the dye TRITC (Xiamen Bioluminor Bio-Technology Co., Ltd.), and the percentage of cells with phagocytosed bacteria was analyzed by flow cytometry (Cytoflex LX, Beckman Coulter).

Cell cytotoxicity assay

Cell cytotoxicity of bacteria was evaluated as the LDH release of RAW-264.7 cells co-cultured with the tested bacteria. Briefly, monolayers of RAW-264.7 cells were grown in DMEM medium without phenol-red with supplementary of 10% fetal bovine serum and were seeded in 96-well flat-bottomed cell culture plates at 2.0  ×  10⁴ cells/well at 37°C in a humidified incubator with 5% CO₂. The cells were co-cultured with PBS washed bacteria for 18 h at an MOI value of 1,000. The LDH of the supernatant was quantified by using a CytoTox96 non-radioactive cytotoxicity kit (Promega, Beijing, China) following the manufacturer’s instructions.

RNA manipulation, real-time RT-PCR

The expression levels of genes were detected by RT-qPCR. The total RNA of different tissues or RAW264.7 cells was extracted by the TRIzol method. The RT-qPCR primers were designed according to the literature (Table S1). The expression of the housekeeping gene β-actin was used for data normalization, and the relative expression levels were estimated by setting the transcript level in mice infected with wild-type as 1. The relative expression of the indicated genes was calculated by the 2^−ΔΔCt method.

Western blot analysis of the toll-like receptor pathway protein expressions

Western blot was performed to assess TLR pathway protein expression for TLR4, MYD88, and NFκB. Briefly, RAW264.7 samples were mixed with RIPA buffer containing protease inhibitor; the extracted protein was measured using BCA Protein Assay Kit (Beyotime Biotechnology, China). Thereafter, 50 µg of the total extracted protein was separated via SDS-PAGE and blotted onto PVDF membranes. PVDF membranes were blocked by incubation in TBS enclosing 3% bovine serum albumin and 0.1% Tween 20 for 1 h at room temperature. PVDF membranes were washed (TBS containing 0.1% Tween 20), and incubated first with a 1:1,000 dilution of the primary antibodies (TLR 4, MYD88, and NFκB p65) for 2 h, and then with a 1:5,000 dilution of the secondary antibody at room temperature. TLR4 [(D8L5W) Rabbit mAb #14358], MyD88 [(D80F5) Rabbit mAb #4283], NFκB p65 [(D14E12) XP Rabbit mAb #8242], reference gene [GAPDH (D16H11) XP Rabbit mAb #5174], goat anti-rabbit immunoglobulin (Ig) G-horseradish peroxidase (HRP) (RS0002) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The chemiluminescence produced was detected with the chemiluminescence scanner (Bio-Rad, USA), and the band intensity was analyzed using the scanner software.

Biofilm formation assays

Twenty-four hours of biofilm formation on polystyrene 96-well plates (Corning, USA) was assessed using crystal violet (Solarbio, China) staining and spectrophotometry under the OD_570nm with a microplate reader (Multiskan FC) as previously described (28). The biomass and structure of the biofilm were also observed by confocal laser scanning microscope (CLSM) (Leica, STELLARIS 5) after CYTO-9 fluorescent staining of the biofilm formed at 24 h.

Statistical analysis

All data from the study were expressed as means ± SD. Analysis of variance (ANOVA) was used to determine the level of significant differences between all groups and Tukey’s honest significant difference for pairwise comparison. Survival analysis was performed using the log-rank (Mantel-Cox) test. P < 0.05 was considered to be statistically significant.

FUNDING

This work was supported by research grants from the National Natural Science Foundation of China (no. 82172328), the Zhejiang Province Natural Science Foundation of China (no. LQ22H200004), and the Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province (2022E10022).

DATA AVAILABILITY

The raw data have been submitted to the SRA database of NCBI (accession numbers SRX19980337, SRX19980338, SRX19980339, SRX19980340, SRX19980341, and SRX19980342).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/iai.00012-24.

Supplemental material. iai.00012-24-s0001.docx.

Isolate resources, Fig. S1, and Table S1.

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