Table 1.

In Vivo Phenotype Analysis of ∆proABC/piaA and ∆fhs/piaA Mutant Strains in Mouse Infection Models

Infection Conditions and Model	Target Organ and (if Applicable) Group	6B Wild Type		∆proABC/piaA		∆fhs/piaA		PBS Control Mice
Infection Conditions and Model	Target Organ and (if Applicable) Group	Log₁₀ cfu/ml	n	Log₁₀ cfu/ml	n	Log₁₀ cfu/ml	n	Log₁₀ cfu/ml	n
Virulence models
Pneumonia	Lungs	5.26 (0.55)	5	3.78 (1.50)^*	5	4.54 (0.35)	5	N/A	—
Pneumonia	Blood	3.30 (0.77)	5	0 (0)^†	5	0 (0)^†	5	N/A	—
Colonization	Nasal wash	4.13 (0.41)	10	3.99 (0.05)	5	3.55 (0.46)	5	N/A	—
Colonization-then-challenge models
6B pneumonia^‡	Lungs	3.70 (1.86)	15	2.51 (2.03)	16	2.40 (3.24)^†	11	3.66 (2.68)	16
	Blood	0 (0)^§	15	0 (0)^§	16	0 (0)^§	11	2.09 (3.89)	16
	BALF	2.00 (2.18)^†	15	2.00 (2.38)	16	0 (1.09)^*	11	2.83 (1.20)	16
6B pneumonia in μMT mice	Lungs, wild-type control mice	4.54 (0.86)	6	4.10 (0.50)	6	3.11 (1.46)	6	N/A	—
	Lungs, μMT mice	4.08 (0.32)	3	4.18 (0.88)	6	4.28 (0.39)	6	N/A	—
	Blood, wild-type control mice	0 (0)	6	0 (0)	6	0 (0)	6	N/A	—
	Blood, μMT mice	3.54 (0.77)^†	3	0 (2.48)	6	3.23 (0.43)^*	6	N/A	—
6B pneumonia in CD4⁺ cell–depleted mice	Lungs, untreated control mice	3.27 (2.91)	6	3.59 (2.89)	6	3.24 (2.73)	6	N/A	—
	Lungs, CD4⁺ cell–depleted mice	3.54 (2.19)	5	1.33 (2.88)	6	3.47 (1.40)	6	N/A	—
	Blood, untreated control mice	0 (0)	6	0 (0)	6	0 (0)	6	N/A	—
	Blood, CD4⁺ cell–depleted mice	0 (0)	5	0 (0)	6	0 (0)	6	N/A	—
6B colonization^‡	Nasal wash	2.71 (1.46)^†	10	1.54 (1.81)^§	10	2.54 (0.94)^*	10	3.88 (0.16)	10
TIGR4 colonization	Nasal wash, untreated control mice	1.00 (1.78)^*	5	1.98 (2.99)^†	6	1.75 (1.66)^†	6	3.84 (0.47)	6
TIGR4 colonization	Nasal wash, CD4⁺ cell–depleted mice	2.52 (0.92)	6	2.90 (1.03)	6	3.38 (0.40)	6	3.36 (0.73)	6

Definition of abbreviations: BALF = BAL fluid; N/A = not applicable; PBS = phosphate-buffered saline; S. pneumoniae = Streptococcus pneumoniae.

For the pneumonia model, colony-forming units were obtained 28 hours after intranasal inoculation with 1 × 10⁷ S. pneumoniae CFU in 50 μl PBS under deep isoflurane anesthesia, and for the colonization model, 7 days after colonization with 1 × 10⁷ cfu in 10 μl PBS under light isoflurane anesthesia (4, 6, 8). For colonization-then-challenge experiments, mice underwent two episodes of colonization (Days 0 and 14) with 1 × 10⁷ cfu in 10 μl PBS under light isoflurane anesthesia of wild-type BHN418 6B, ∆proABC/piaA, or ∆fhs/piaA S. pneumoniae strains or sham colonization with PBS before challenge between Days 30 and 42. For these, the data presented are pooled from two experiments except the colonization-with-∆proABC/piaA-and-then-challenge experiment, which includes pooled data from three experiments. The μMT^−/− mice were in the C57B/6J background (kind gift from Claudia Mauri, University College London); all other experiments used CD1 mice aged 4–8 weeks. CD4⁺ cells were depleted by intraperitoneal injection of 250 μg anti-CD4 mAb (GK 1.5; BioxCell) 48 and 24 hours before S. pneumoniae challenge (6); flow cytometry confirmed >99% depletion of splenic CD4⁺ cells (data not shown). Data are presented as the median of log₁₀ cfu/ml (interquartile range) recovered from target organs after infection with wild-type, ∆proABC/piaA, ∆fhs/piaA, or PBS control mice. P values were obtained using Kruskal-Wallis tests with Dunn’s post hoc test comparing groups with the wild-type 6B strain (for virulence models) or the PBS sham-colonized group (for colonization-then-challenge data). The Mann-Whitney test was used for experiments with μMT^−/− mice and CD4⁺ cell–depleted mice, comparing wild-type/untreated mouse data with μMT^−/−/CD4⁺ cell–depleted mouse data, respectively, for each target organ and S. pneumoniae strain combination.

P < 0.01.

^{^†}

P < 0.05.

^{^‡}

Combined data from two repeated experiments.

^{^§}

P < 0.001.